Novel microRNA prosurvival cocktail for improving engraftment and function of cardiac progenitor cell transplantation

Abstract

Background: Although stem cell therapy has provided a promising treatment for myocardial infarction, the low survival of the transplanted cells in the infarcted myocardium is possibly a primary reason for failure of long-term improvement. Therefore, the development of novel prosurvival strategies to boost stem cell survival will be of significant benefit to this field.

Methods and results: Cardiac progenitor cells (CPCs) were isolated from transgenic mice, which constitutively express firefly luciferase and green fluorescent protein. The CPCs were transduced with individual lentivirus carrying the precursor of miR-21, miR-24, and miR-221, a cocktail of these 3 microRNA precursors, or green fluorescent protein as a control. After challenge in serum free medium, CPCs treated with the 3 microRNA cocktail showed significantly higher viability compared with untreated CPCs. After intramuscular and intramyocardial injections, in vivo bioluminescence imaging showed that microRNA cocktail-treated CPCs survived significantly longer after transplantation. After left anterior descending artery ligation, microRNA cocktail-treated CPCs boost the therapeutic efficacy in terms of functional recovery. Histological analysis confirmed increased myocardial wall thickness and CPC engraftment in the myocardium with the microRNA cocktail. Finally, we used bioinformatics analysis and experimental validation assays to show that Bim, a critical apoptotic activator, is an important target gene of the microRNA cocktail, which collectively can bind to the 3'UTR region of Bim and suppress its expression.

Conclusions: We have demonstrated that a microRNA prosurvival cocktail (miR-21, miR-24, and miR-221) can improve the engraftment of transplanted cardiac progenitor cells and therapeutic efficacy for treatment of ischemic heart disease.

Figure 1. Characterization and differentiation of CPCs

(A) The morphology of isolated CPCs growing on gelatin coated dish. (B) Flow cytometric analysis of purified Sca-1⁺ CPCs population. CPCs isolated from transgenic mice express robust (C) firefly luciferase and (D) GFP expression. After culturing in induction medium, differentiated CPCs stained positively with (E) connexin 43, (F) cardiac Troponin T, (G) myocyte-specific enhancer factor 2C, and (H) α-smooth muscle actin. (I) Cell morphology of CPC-derived endothelial cells. (J) These CPC-derived endothelial cells can uptake DiI-Ac-LDL. (K) These CPC-derived endothelial cells can form tube-like structure on Matrigel angiogenesis assay. (L) In contrast, the undifferentiated CPCs cannot form tube-like structure.

Figure 2. Screening of miRNAs for improving cell viability

After CPCs were transduced with several individual miRNAs or miRNAs cluster candidate, cells were exposed to serum free medium. Cell viability was measured after 24 hours of serum free challenge. Note the CPC viability is significantly higher in the presence of the miRNA cocktail (miR-21, miR24, and miR221). One-way ANOVA analysis was used.

Figure 3. Correlation between CPC numbers and bioluminescence signals

(A) CPCs were seeded into 6-well plate with increasing cell numbers. (B) Assessment of bioluminescence signals showed a robust linear correlation (R²=0.99) between the cell number and Fluc expression, which is crucial for accurate tracking of cell survival by in vivo imaging. Pearson’s correlation was used. Each data point is from individual observation.

Figure 4. Direct comparison of cell survival in vivo by bioluminescence imaging

(A) Representative bioluminescence images of animals injected with CPCs alone (left leg) vs. CPCs treated with miRNA cocktail (right leg). Note this is in the setting of an uninjured hindlimb injection model. (B) Quantitative analysis of longitudinal BLI signal following intramuscular hindlimb injection. Two-way RMANOVA was used. (C) Following myocardial infarction, adult mice were injected intramyocardially with CPCs alone vs. CPCs treated with miRNA cocktail. (D) Quantitative analysis of BLI signals demonstrated that CPCs treated with miRNA cocktail showed significantly better survival at all time points. 2-way ANOVA was used for statistical analysis; two-way RMANOVA was used.

Figure 4. Direct comparison of cell survival in vivo by bioluminescence imaging

(A) Representative bioluminescence images of animals injected with CPCs alone (left leg) vs. CPCs treated with miRNA cocktail (right leg). Note this is in the setting of an uninjured hindlimb injection model. (B) Quantitative analysis of longitudinal BLI signal following intramuscular hindlimb injection. Two-way RMANOVA was used. (C) Following myocardial infarction, adult mice were injected intramyocardially with CPCs alone vs. CPCs treated with miRNA cocktail. (D) Quantitative analysis of BLI signals demonstrated that CPCs treated with miRNA cocktail showed significantly better survival at all time points. 2-way ANOVA was used for statistical analysis; two-way RMANOVA was used.

Figure 5. Evaluation of cardiac functional recovery after myocardial infarction

(A) Representative echocardiographic images of healthy heart vs. infarcted hearts receiving PBS, CPCs alone, and CPCs treated with miRNA cocktail at day 28. (B) Comparison of fractional shortening (FS) among the 3 groups. Compared with PBS control, CPCs treated with miRNA cocktail group had significantly higher FS (P<0.05). The CPC alone group only showed a positive trend toward improvement (P=0.18). Two-way RMANOVA was used for statistical analysis. (C) Representative Masson trichrome staining of explanted heart showed increased wall thickness for CPCs treated with miRNA cocktail group, confirming the positive echocardiographic data. * represents left ventricle. (D) Engraftment of transplanted CPCs was confirmed by GFP staining. As expected, CPCs treated with miRNA cocktail group showed significantly higher engraftment compared with CPC alone group. Green: GPF; Red: cTnT; Blue: DAPI.

Figure 6. Schematic diagram for constitutive reporter vector and confirmation of the target genes of miRNAs

(A) For luciferase assay, the segment of Bim 3’ UTR containing miRNA binding site was amplified by PCR and inserted downstream of Fluc driven by SV40 promoter. (B) Dual luciferase assay indicated the luciferase activity of the reporters could be suppressed by relative miRNAs, except for reporter 221_1. One-way ANOVA was used. (C) Western blot showed the isoforms of Bim in CPCs could be inhibited by miRNA cocktails endogenously.