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

Abstract

Stromal cell-derived factor-1alpha (SDF-1alpha) 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-1alpha 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-1alpha into a collagen substrate to create a microenvironment favoring stem cell homing. Local release of SDF-1alpha 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-1alpha 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 microg/cm(2), 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 x 10(5) RLU/mg protein. GAC allowed the sustained gene release up to 60 days. GAC mediated SDF-1alpha 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.

Fig 6

In vitro statistic analysis of migration and homing of CD117⁺ cells. (A) Flow chamber employed to simulate the circulating environment. (B) Representative FACS histogram of CD117⁺ cells purity assay. (C) The number of CD117⁺ cells on the GAC-coated area normalized by the number of COS7 cells. (D) EGFP expression of COS7 cells cultured on the GAC (PEI/pEGFP-N3-SDF-1α/collagen)-coated area, (E) a CD117⁺ cell labelled with CellTracker Probe-Red CMTPX. (F) Merged picture of fluorescence and phase-contrast microscopic picture and (G) enlarged 3D-picture that indicates a homed CD117⁺ cell on a transfected COS7 cell. (Bars in D–F = 30 μm; Bar in G = 10 μm).

Fig 1

Characterization of GAC. Plasmid DNA (pREP4-Luc) retardation by (A) PEI at various N/P ratios and (B) collagen at various C/D ratios. (C) GAC (PEI/pEGFP-N3-SDF-1α/collagen) complexes size influenced by N/P and C/D ratios. (D) Released speed of PEI/DNA (pcDNA3.1-LacZ) complex from collagen substrate. The results were expressed as average ± standard deviation (N= 6).

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 (μg/cm²)) were varied. Collagen dosage was constant (10 μg/cm²). The result was given as average ± standard deviation (N= 6). One way-ANOVA analysis was carried out to determine the optimal parameters corresponding to the highest cell viability for GAC group (*P < 0.05, **P < 0.01). Cells growing on GAC (7.5 μgDNA/cm², N/P= 2) have the highest viability comparing with cells on other GACs (P < 0.05).

Fig 3

Luciferase expression of COS7 cells transfected by PEI/DNA (pREP4-Luc) complexes at the absence (collagen free) and presence of collagen (GAC). N/P ratios and DNA dosages (expressed as DNA amount in unit area (μg/cm²)) were varied. Collagen dosage was constant (10 μg/cm²). The result was given as average ± standard deviation (N= 6). One way-ANOVA analysis was carried out to determine the optimal parameters corresponding to the highest transfection efficiency for GAC group (*P < 0.05, **P < 0.01). Cells growing on GAC (10.0 μgDNA/cm², N/P= 4) have the highest transfection efficiency comparing with cells on other GACs (P < 0.05).

Fig 4

EGFP expression of COS7 cells transfected by GAC (PEI/pEGFP-N3-SDF-1α/collagen) assembled with optimized parameters (DNA dosage 10 μg/cm², N/P ratio 4 and C/D ratio 1). (A) Cells expressing EGFP, (B) phase contrast image of cells, (C) merged image. (Bar = 100 μm).

Fig 5

Localized gene delivery mediated by GAC. X-gal staining of pcDNA3.1-LacZ transfected COS7 cells appeared blue in (A) complete view and (B) enlarged view. Transfected COS7 cells locally expressed EGFP in designed areas. (C) Fluorescence microscopic picture, (D) phase-contrast microscopic picture and (E) merged picture. The dotted lines indicate the border of the GAC-coated and uncoated areas. (Bar in A = 5 mm; Bars in B–E = 100 μm).

Fig 7

In vivo co-expression of EGFP and SDF-1α induced by GAC (PEI/pEGFP-N3-SDF-1α/collagen) was evaluated by triple staining of EGFP, SDF-1α 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-1α staining and (C, G) nuclei staining and phase-contrast microscopic pictures (D, H). (Bar = 100 μm).

Fig 8

The in vivo up-regulation of CD117 expression induced by GAC was evaluated by RT-PCR. The group transplanted with naked membrane was used as control group. The result was expressed as the fold change over the average value of the control group, which was set as 1. (A) Representative bands visualized under UV illuminator. (B) CD117 mRNA levels were given with respect to control group (naked membrane, N= 6) and expressed as average ± standard deviation (pEGFP-N3 group: N= 6; pEGFP-N3-SDF-1α group: N= 8). The CD117⁺ cells were identified by immunostaining in the tissues transplanted with GAC-coated membranes, as shown in (C) fluorescence microscopic picture (Bar = 30 μm) and (D) enlarged picture (Bar = 10 μm).

Fig 9

The inflammation induced by GAC (PEI/pEGFP-N3-SDF-1α/collagen, N/P = 4, C/D = 1) was evaluated with H&E staining. GAC was coated onto the mixed cellulose ester membrane with 40 μg DNA/membrane (3 mm × 4 mm), and then the membrane was transplanted into the quadriceps of mice. The tissues transplanted with naked membrane were used as control and harvested 3 days after transplantation (A). The tissues transplanted with GAC-coated membrane were harvested, respectively, at days 3 (B), 7 (C) and 14 (D) after transplantation. (Bar = 200 μm). The arrows indicate the naked membrane (A) and GACs-coated membranes (B, C, D).