Figure 1.
Characterization of rat bone marrow MSCs (α-MEM).
(A) Isolated MSCs were round and small at 2 days after isolation. (B) After three passages, MSCs became larger and multiangular. (C) Before use, MSCs were mostly found in the clostridial form. (D, E, F, G) Fluorescent immunocytostaining showed that cultivated cells expressed CD29, strongly expressed CD105 and did not express CD14 and CD45. (H) Osteocyte lineage differentiation potential was determined by Alizarin Red S staining. (I) Oil Red O staining indicated the accumulation of oil droplets in cells that differentiated to adipocytes.
Figure 2.
Transfection efficiency in MSCs.
(A) Staining for fluorescence microscopy showed that MSCs expressed eGFP (original magnification 400× and 200×). (B) Western blot analysis was performed to detect CXCR4 protein expression. β-actin was used as protein loading control. Analysis of CXCR4 levels by histogram indicated that protein expression was up-regulated in MSCsCXCR4/GFP(★P<0.05 vs MSCsnative) and down-regulated in MSCsshCXCR4/GFP (▵P<0.05 vs. MSCsnative). (C) Semi-quantitative RT-PCR was used to analyze CXCR4 mRNA levels in MSCs. GAPDH was used as RNA loading control. (★▵P<0.05 vs. MSCsnative).
Figure 3.
Regulation of CXCR4 expression affects the proliferation of MSCs in vitro.
(A) Viability was decreased in MSCsshCXCR4/GFP and increased in MSCsCXCR4/GFP compared to MSCsGFP or MSCsnative (control), as measured by the MTT assay (Mean ± SD; n = 6; P<0.05 from NO. 4 to 5 time point). (B) All cell nuclei exhibited blue fluorescent Hoechst 33342 staining, and EdU labeling indicated replicating cells. In MSCsCXCR4, an increased number of cells exhibited red fluorescent EdU labeling following CXCR4 overexpression. In MSCsshCXCR4/GFP, fewer cells exhibited red fluorescence, indicating that EdU labeling decreased with CXCR4 knockdown. (C) The percentages of red fluorescent cells for different MSC groups are indicated in the histogram. There were more replicating cells for MSCsCXCR4/GFP than the control group. ★P<0.05 vs. control, and ▵P<0.01 vs. MSCsshCXCR4/GFP.
Figure 4.
Antibody-based protein array system.
Positive controls are located in the upper left-hand corner (four spots) and lower right-hand corner (two spots) of each membrane. (A) MSCsCXCR4/GFP. (B) MSCsshCXCR4/GFP. (C) MSCsGFP. (D) MSCsnative. Down-regulated or up-regulated proteins are indicated with arrowheads. Histogram shows protein array results. (E) down-regulated proteins. (F) up-regulated proteins. ★P<0.05 vs. MSCsGFP.
Figure 5.
Injury in transplanted organs.
(A, B) Morphology analysis showed apparent injury in transplanted organs. (C) SDF-1 protein was up-regulated in transplanted kidneys. The kidney tissue lysates from recipient rats were analyzed by ELISA to determine SDF-1 protein expression at 5 time points after transplant surgery. The histogram indicates that SDF-1 expression was up-regulated in transplanted kidneys at different time points post-surgery time. ★P<0.05 vs. normal kidney. (D) Pathology score analysis showed a disparity. Normal kidneys were used as controls (methods not shown). ★P<0.05 vs. control.
Figure 6.
The role of the SDF-1-CXCR4 pathway in the homing of MSCs.
(A) Kidney uptake of eGFP-labeled MSCs was measured by fluorescence microscopy 48 h after systemic administration (a: MSCsCXCR4/GFP, b: MSCsGFP, c: MSCsshCXCR4/GFP). Labeled cells were detected surrounding the glomerulus (arrows). (B and C) Chemotaxis into transplanted kidneys was measured by RT-PCR andWestern blot. Recipients treated with MSCsGFP were used as control. ★P<0.05 vs. control. (D) The expression of eGFP in different organs from recipient rats was measured 48 h after infusion.
Figure 7.
Transplantation of MSCs was renoprotective.
The effects of the SDF-1-CXCR4 pathway on the therapeutic efficacy of MSCs for the treatment of injury in transplanted kidneys was evaluated by measuring serum urea, serum creatinine, pathology scores, and immunohistochemisty staining. BUN and Scr levels were measured in recipients that received MSCs or PBS 12 h and 72 h after the surgery. There were no difference between each group 12 h after the surgery (A and B), but there were significant diversity 72 h after the surgery (C and D). ★P<0.05 vs. MSCsCXCR4/GFP; ▵P<0.05 vs. PBS. (E) HSK in transplanted kidneys that received different MSCs or PBS were calculated 72 h after the surgery (n = 8 per group). ★P<0.05 vs.MSCsCXCR4/GFP ; ▵P<0.05 vs. PBS. (F) Expression of CD25, FOXP3, or CD45 in the renal interstitium of native renal tissues or at different MSCs-treated groups after renal transplantation by immunohistochemistry. a, b, c: Paraffin-embedded sections of normal renal tissues showing weaker expression of CD25, or FOXP3, or CD45 [diaminobenzidine (DAB), original magnification 200×]. P<0.05 vs. transplanted kidney. d∼p: Paraffin-embedded sections of transplanted kidney exhibiting positive expression of CD25, or FOXP3, or CD45 in the renal interstitium (DAB, original magnification 200×). The expression of CD45,CD25 and FOXP3 were hyper in MSCsCXCR4/GFP-treated group.★ P<0.05 vs. MSCsCXCR4/GFP-treated group. ▵P<0.05 vs. PBS-treated group. Scale bars represent 50 µm.