CXCR4 Overexpression in Human Adipose Tissue-Derived Stem Cells Improves Homing and Engraftment in an Animal Limb Ischemia Model

Abstract

We investigated the effects of transplantation of CXCR4-overexpressing adipose tissue-derived stem cells (ADSCs) into a mouse diabetic hindlimb ischemia model on homing and engraftment as early as 48 h after transplant. CXCR4-overexpressing ADSCs were intramuscularly or intravenously injected into diabetic mice with hindlimb ischemia. After 48 h, muscle tissues in the femur and tibia were collected, and the CXCR4 expression pattern was analyzed by immunofluorescence staining. The homing and engraftment of transplanted CXCR4-overexpressing ADSCs into the ischemic area were significantly increased, and intravenous (systemic) injection resulted in the more effective delivery of stem cells to the target site 48 h posttransplantation. Furthermore, CXCR4-overexpressing ADSCs more efficiently contributed to long-term engraftment and muscle tissue regeneration than normal ADSCs in a limb ischemia model. In addition, the homing and engraftment of ADSCs were correlated with the CXCR4 transfection efficiency. These results demonstrated that enhanced CXCR4 signaling could significantly improve the early homing and engraftment of ADSCs into ischemic areas as well as the long-term engraftment and ultimate muscle tissue regeneration.

Figure 1

The cloning map for green fluorescent protein (GFP) and human C-X-C chemokine receptor type 4 (hCXCR4) into pWPI lentiviral vector. The hCXCR4 complementary DNA (cDNA) (NdeI–MluI 1.0-kb fragment) was cloned into the pWPI lentiviral vector between the EF1-α promoter and the EMCV IRES-GFP. Abbreviations: Amp^r, ampicillin resistance gene; F1 ori, origin of replication from f1 phage; pGEM, plasmid for cloning CXCR4 gene; Bgl2, restriction endonuclease from Bacillus globigii; Spe1, restriction endonuclease from Sphaerotilus species; LTR, long terminal repeat; pWPI-MCS, plasmid from Addgene (plasmid #12254) with multicloning site (MCS); Mlu, restriction endonuclease from Micorcoccus luteus; Nde1, restriction endonuclease from Neisseria denitrficans; BamH1, restriction endonuclease H1 from Bacillus amyloliquefaciens; EF1-α, elongation factor 1-α; EMCV IRES, encephalomyocarditis virus internal ribosomal entry site.

Figure 2

Flow cytometric analysis of GFP and CXCR4 expression in transfected adipose tissue-derived stem cells (ADSCs). Each group of ADSCs was modified with the GFP vector (A) or GFP/CXCR4 vector (B, C). Modified ADSCs were stained with PE-conjugated anti-CXCR4 antibody and analyzed. ADSCs with only the GFP vector (B) were positive for GFP (99.83%), and CXCR4-overexpressing ADSCs (C) were positive for GFP (96.04%) and CXCR4 (99.92%).

Figure 3

Detection of CXCR4⁺ and GFP⁺ by immunofluorescent (IF) staining in diabetic mouse ischemic muscle after 48 h posttransplantation. IF staining was performed for GFP (FITC, green) and CXCR4 [cyanine3 (Cy3), red], and nuclei were stained with DNA-binding dye [4′,6-diamidino-2 phenylindole dihydrochloride (DAPI), blue]. GFP and CXCR4 expression levels were increased significantly in the intravenous (IV) group of CXCR4-overexpressing ADSCs compared to other experimental groups after 48 h. Overall CXCR4-overexpressing ADSCs showed much higher engraftment than GFP groups. Damaged site in the diabetic ischemic muscle was analyzed by Masson's trichrome (MT) staining. DM, diabetes mellitus; i.m., intramuscular; i.v., intravenous. Original magnification: 200×, scale bar: 50 μM.

Figure 4

Quantification of fluorescence intensity of Figure 3 was done using ImageJ software (ImageJ for Windows, Version 1.50i), and statistical significance was analyzed using SPSS Statistics for Windows, Version 23.0 (IBM Corp.). All results were statistically significant (∗∗p < 0.01, ∗∗∗p < 0.001).

Figure 5

Detection of CXCR4⁺ and GFP⁺ cells by IF staining in mouse ischemic muscle after 96 h posttransplantation. IF staining was performed for GFP (green) and CXCR4 (Cy3, red), and nuclei were stained with DNA-binding dye (DAPI, blue). GFP and CXCR4 expression levels were similar both in the IM and IV group of GFP- and CXCR4-overexpressing ADSCs. In this set of experiment, CXCR4 transfection efficiency was less than one fourth of GFP's. Because of the low transfection efficiencies, the CXCR4-transfected cells were not dominant in the damaged muscle site. The damaged site in the diabetic hindlimb ischemic muscle was analyzed by MT staining. i.m., intramuscular; i.v., intravenous. Original magnification: 200×, scale bar: 50 μM.

Figure 6

Quantification of fluorescence intensity of Figure 5 was done using ImageJ software (ImageJ for Windows, Version 1.50i), and statistical significance was analyzed using SPSS Statistics for Windows, Version 23.0 (IBM Corp.). All results were statistically significant (∗p < 0.05, ∗∗∗p < 0.001).

Figure 7

Detection of CXCR4⁺ and GFP⁺ cells by IF staining in canine ischemic muscle after 8 weeks posttransplantation. IF staining was performed for GFP (green) and CXCR4 (Cy3, red), and nuclei were stained with DNA-binding dye (DAPI, blue). After 8 weeks, GFP and CXCR4 expression levels were detected in the IM injection group of CXCR4-overexpressing ADSCs. The damaged site in the canine hindlimb ischemic muscle was analyzed by MT staining. i.m., intramuscular. Original magnification: 200×, scale bar: 50 μM.

Figure 8

Quantification of fluorescence intensity of Figure 7 was done using ImageJ software (ImageJ for Windows, Version 1.50i), and statistical significance was analyzed using SPSS Statistics for Windows, Version 23.0 (IBM Corp.). All results were statistically significant (∗p < 0.05, ∗∗p < 0.01).

Figure 9

Angiography results for confirmation of angiogenesis effect. Eight weeks after cell transplantation, the angiograms of the limbs were obtained. GFP- and CXCR4-overexpressing ADSC-treated limbs showed improved vascularity compared to the control ischemia limbs. The CXCR4-overexpressing treated limbs exhibited the most abundant vascularity. Ischemia only: normal saline, GFP: 1 × 10⁷ ADSCs, CXCR4: 1 × 10⁷ CXCR4-overexpressing ADSCs. Scale bar is marked at the right side.