Transplantation of CRISPRa system engineered IL10-overexpressing bone marrow-derived mesenchymal stem cells for the treatment of myocardial infarction in diabetic mice

Fig. 1. CRISPR/dCas9 activation (CRISPRa) system mediated overexpression of IL10 in Bone Marrow-Derived Mesenchymal Stem Cells (BM-MSCs). a Immunophenotypic profile of BM-MSCs. Flow cytometry histograms after three passages show the expression of selected surface molecules (including CD29, CD44, CD90, CD34 and CD45). Cells were stained with FITC-labeled antibodies or isotype control antibody. The BM-MSCs were positive for CD29, CD44, and CD90 but negative for CD34 and CD45. b Establishment of streptozotocin-induced diabetic model. 6–8-week-old BALB/c mice were intraperitoneal (i.p.) injection with Streptozotocin (STZ) (40 mg/kg) for 5 consecutive days. The IL-10 expressions in mRNA level (c) and protein level (d) of BM-MSCs isolated from healthy mice and streptozotocin (STZ)-induced diabetic mice was examined by real-time PCR (RT-PCR) and Western blotting, respectively. Data represent means ± SD. ***p < 0.001, n = 3. e Production of IL-10 by BM-MSCs and diabetic BM-MSCs. The IL-10 levels in the supernatant were measured by enzyme-linked immunosorbent assay (ELISA). Data represent means ± SD. **p < 0.005, n = 3. f Schematic diagram of CRISPR/dCas9 activation plasmids. The engineered…

Fig. 2. BM-MSCs-pCas9/gIL10 transplantation induces increased expression of IL-10 in diabetic mice with myocardial infarction. a Schematic depicts the experiment protocol. BM-MSCs were isolated and cultured, and then transfected with pCas9/dgCtrl or pCas9/dgIL10. Mice models of diabetic MI were established via coronary artery ligation. STZ-induced diabetic mice were injected with BM-MSCs-pCas9/dgCtrl, pCas9/dgIL10 or BM-MSCs pCas9/dgIL10 at 2 sites near the border zone of infarction (medial and lateral zones) 1 h post myocardial infarction (MI) accomplishment, and 7 days later the mice were subjected to examination. b In vivo optical bioluminescence imaging (BLI). Luciferase gene was inserted into plasmids, and BLI was detected using the Xenogen In Vivo Imaging System 1-weeks post transplantation by intravenously injecting luciferase substrate. Four groups were treated as follows: Control (PBS only), BM-MSCs-pCas9/gCtrl (BM-MSCS transfected with control CRISPR/Cas9 plasmids), pCas9/gIL10 (direct injection of CRISPR/Cas9 plasmids express IL-10), and BM-MSCs-pCas9/gIL10 (BM-MSCS transfected with control CRISPR/Cas9 plasmids express IL10). The number of cells was 2*106, and the mass of…

Fig. 3. Transplantation of BM-MSCs-pCas9/gIL10 suppresses infiltration of inflammatory cells and expression of proinflammatory cytokines in the myocardium. a Representative fluorescent micrographs show the presence of inflammatory cells (CD68-positive, green fluorescence) peri-infarct area in the control MI heart or hearts treated with BM-MSCs pCas9/gCtrl, pCas9/gIL10 or BM-MSCs pCas9/gIL10. The examination was performed 1-week post transplantation. Scale bar was 20 μm. b Flow cytometry analysis of infiltration of CD11b+CD68+ cells peri-infarct area in the heart. Tissues from control MI heart or hearts treated with BM-MSCs-pCas9/gCtrl, pCas9/gIL10 or BM-MSCs-pCas9/gIL10 were digested into single cells stained with FITC labeled CD11b and APC labeled CD68 antibodies, and then examined by flow cytometry. c Bar graph shows quantitative analysis of infiltrating CD68-positive cells. Data represent means ± SD. **p < 0.005, n = 8. d-g Quantitative analysis of mRNA expression of proinflammatory cytokines and chemokines (IL-1β, TNF-α, IL-6 and MCP-1) in the border zone of LV infarct of LV infarct at 1-week post-MI. mRNA expression normalized to GAPDH expression. Data represent means ± SD. *p <…

Fig. 4. Transplantation of BM-MSCs pCas9/gIL10 improves cardiac functional recovery in mice model of diabetic MI. a Representative echocardiographic images 4 weeks after MI and treated with BM-MSCs-pCas9/gCtrl, pCas9/gIL10 or BM-MSCs-pCas9/gIL10. b Left ventricular ejection fraction (LVEF). Data represent means ± SD. *p < 0.05, **p < 0.005, n = 8. c-e Hemodynamic analyses including +dp/dtmax, −dp/dtmax, and LV end diastolic pressure (LVEDP). Data represent means ± SD. *p < 0.05, **p < 0.005, n = 8

Fig. 5. Transplantation of BM-MSCs-pCas9/gIL10 alleviates cardiac injury, decreases apoptosis of cardiac cells and increases angiogenesis in mice model of diabetic myocardial infarction. a Representative Masson’s trichrome–stained myocardial sections in the control MI heart or hearts treated with BM-MSCs pCas9/gCtrl, pCas9/gIL10 or BM-MSCs pCas9/gIL10. Blue, scar tissue; red, viable myocardium. b Quantitation of infarct size in the four indicated experimental groups. Data represent means ± SD. **p < 0.005, n = 8. c Representative fluorescent micrographs showing the presence of TUNEL+ (terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick end labeling–positive) apoptotic cells (green) in the border zone of LV infarct. Nuclei were stained by DAPI (blue). d Quantification of percentage of apoptotic cells in the four indicated experimental groups. Data represent means ± SD. **p < 0.005, n = 8. e Angiogenesis was inspected using von Willebrand factor (vWF) staining in heart sections from different groups 4 weeks after MI and transplantation. Tissue sections were stained primary rabbit anti-vWF antibody and secondary goat anti-Rabbit IgG antibody labeled with Alexa…