CCND2 Overexpression Enhances the Regenerative Potency of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes: Remuscularization of Injured Ventricle

Abstract

Rationale: The effectiveness of transplanted, human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) for treatment of ischemic myocardial injury is limited by the exceptionally low engraftment rate.

Objective: To determine whether overexpression of the cell cycle activator CCND2 (cyclin D2) in hiPSC-CMs can increase the graft size and improve myocardial recovery in a mouse model of myocardial infarction by increasing the proliferation of grafted cells.

Methods and results: Human CCND2 was delivered to hiPSCs via lentiviral-mediated gene transfection. In cultured cells, markers for cell cycle activation and proliferation were ≈3- to 7-folds higher in CCND2-overexpressing hiPSC-CMs (hiPSC-CCND2^OECMs) than in hiPSC-CMs with normal levels of CCND2 (hiPSC-CCND2^WTCMs; P<0.01). The pluripotent genes (Oct 4, Sox2, and Nanog) decrease to minimal levels and undetectable levels at day 1 and 10 after differentiating to CMs. In the mouse myocardial infarction model, cardiac function, infarct size, and the number of engrafted cells were similar at week 1 after treatment with hiPSC-CCND2^OECMs or hiPSC-CCND2^WTCMs but was about tripled in hiPSC-CCND2^OECM-treated than in hiPSC-CCND2^WTCM-treated animals at week 4 (P<0.01). The cardiac function and infarct size were significantly better in both cell treatment groups' hearts than in control hearts, which was most prominent in hiPSC-CCND2^OECM-treated animals (P<0.05, each). No tumor formation was observed in any hearts.

Conclusions: CCND2 overexpression activates cell cycle progression in hiPSC-CMs that results in a significant enhanced potency for myocardial repair as evidenced by remuscularization of injured myocardium. This left ventricular muscle regeneration and increased angiogenesis in border zone are accompanied by a significant improvement of left ventricular chamber function.

Keywords: cell cycle; cyclin D2; induced pluripotent stem cells; myocardial infarction; regeneration.

Figure 1. CCND2 overexpression promotes cell-cycle progression and telomerase activity in hiPSC-CMs

hiPSC-CCND2^WTCMs (CCND2^WT) and hiPSC-CCND2^OECMs (CCND2^OE) were cultured for 4 weeks after initiation of cardiomyocyte differentiation, and then immunofluorescently stained for the presence of hcTnT; nuclei were identified via Nkx2.5 expression or DAPI staining. (A) Proliferation was evaluated via immunofluorescence analyses of Ki67 expression in hiPSC-CCND2^WTCMs and hiPSC-CCND2^OECMs and quantified as the percentage of positively stained cells. Bar = 20 μm. (B) S-phase cells were identified via immunofluorescence analyses of Brdu incorporation in hiPSC-CCND2^WTCMs and in two clones of hiPSC-CCND2^OECMs and quantified as the percentage of positively stained cells. Bar = 50 μm. (C) M-phase cells were identified via analyses of PH3 expression and quantified as the percentage of positively stained cells. Bar = 20 μm. (D) Cells undergoing cytokinesis were identified via Aurora B expression and quantified as the percentage of positively stained cells. Bar = 20 μm. (E) Apoptotic cells were identified via TUNEL staining and quantified as the percentage of positively stained cells. Bar = 20 μm. *P<0.05 versus CCND2^WT. (F) (i) hiPSC-CCND2^WTCMs (CCND2^WT) and hiPSC-CCND2^OECMs (CCND2^OE) were cultured for 24 weeks; S-phase cells were identified at the indicated time points via immunofluorescence analyses of Brdu incorporation and quantified as the percentage of positively stained cells. *P<0.05 between CCND2^OE and CCND2^WT at each timepoint. ^#P<0.05 versus CCND2^WT at 4 weeks. ^†P<0.05 versus CCND2^WT at 8 weeks. ^‡P<0.05 versus CCND2^WT at 12 weeks. ^¥P<0.05 versus CCND2^OE at 4 weeks. ^§P<0.05 versus CCND2^OE at 8 weeks. (ii) Telomerase activity was evaluated with a non-denaturing polyacrylamide gel and (ii) quantified at the indicated time points via densitometry analysis. *P<0.05 between CCND2^OE and CCND2^WT at each timepoint. ^#P<0.05 versus CCND2^WT at 1 week. ^¥P<0.05 versus CCND2^OE at 1 week. All experiments were repeated for twice (n=3). A minimal of 900 cells from each group were counted.

Figure 2. hiPSC-CCND2^OECMs are more potent than hiPSC-CCND2^WTCMs for the treatment of MI in mice

Animals were treated with intramyocardial injections of hiPSC-CCND2^OECMs (CCND2^OE), hiPSC-CCND2^WTCMs (CCND2^WT), or PBS after surgically induced MI; a fourth group of animals (Sham) underwent sham surgery. (A) Echocardiographic assessments of left-ventricular (A) ejection fractions (EF) were performed before MI induction (Pre-MI) and 1 and 4 weeks afterward. Echocardiography data was presented as absolute numbers (left panel), and was normalized to individual pre-MI baseline values (right panel). (B-C) Sections from the left ventricles of mice sacrificed (B) 1 week and (C) 4 weeks after MI induction were stained with fast green to identify functional myocardium and with sirius red to identify scar tissue; then, infarct size was quantified as the ratio of the scar area to the total surface area and expressed as a percentage. (D-E) The hypertrophic response to MI injury was evaluated by determining (D) the heart weight:body weight ratio and (E) the minimal diameter of cardiomyocyte fibers in the left ventricles of animals sacrificed 1 and 4 weeks after MI induction. *P<0.05 versus Sham, ^#P<0.05 versus PBS, ⁺P<0.05 versus CCND2^WT.

Figure 3. More hiPSC-CCND2^OECMs than hiPSC-CCND2^WTCMs are present in infarcted mouse hearts at week 4 after transplantation

The transplanted cells carried a luciferase reporter plasmid and were of human origin; thus, engraftment was evaluated via (A-E) bioluminescence imaging (BLI) and (F-G) by identifying cells that expressed the human variant of cardiac troponin T (hcTnT) and human nuclear antigen (HNA). (A) A standard curve was generated from BLI measurements of known quantities of hiPSC-CMs. (B) One and 4 weeks after MI induction and treatment with intramyocardial injections of hiPSC-CCND2^OECMs (CCND2^OE) or hiPSC-CCND2^WTCMs (CCND2^WT), the mice were injected with luciferin, and BLI images were collected 10 minutes later. (C) BLI signal intensity was compared to the standard curve to determine (D) the number of engrafted cells; then, (E) the engraftment rate was calculated by dividing the number of engrafted cells by the total number of cells administered and expressed as a percentage. *P<0.05 versus Sham, ^#P<0.05 versus PBS, ⁺P<0.05 versus CCND2^WT. (F) (i) Heart sections from mice that had been sacrificed 1 and 4 weeks after MI induction and treatment with hiPSC-CCND2^WTCMs or hiPSC-CCND2^OECMs were stained for the presence of hcTnT and HNA; nuclei were counterstained with DAPI (10× images: bar=100 μm, 40× images: bar=20 μm). (ii) The engraftment rate was calculated by dividing the number of cells that expressed both hcTnT and HNA by the total number of cells administered and expressed as a percentage. *P<0.05 versus CCND2^WT. (iii) Sections were stained with wheat germ agglutinin (WGA) to define the cell border and with antibodies against HNA and hcTnT; then, the proportion of HNA-positive cells that also co-expressed hcTnT was determined and expressed as a percentage. Panels C-E: SHAM (n=8), PBS (n=10), CCND2^WT (n=10), and CCND2^OE (n=10) at week 1; SHAM (n=8), PBS (n=9), CCND2^WT (n=9), and CCND2^OE (n=9) at week 4. Panel G-H: n=5 animals per treatment group at week 1 and 4; 32–40 sections per animal were evaluated, and 1–8 high power of fields per section were assessed.

Figure 4. Cell-cycle activity and proliferation are greater in hiPSC-CCND2^OECMs than in hiPSC-CCND2^WTCMs at week 4 after transplantation

(A–C) Engrafted hiPSC-CCND2^WTCMs (CCND2^WT) and hiPSC-CCND2^OECMs (CCND2^OE) were identified in mouse hearts at week 1 and week 4 after MI induction and treatment via immunofluorescent staining for the expression of hcTnT and hNkx2.5; then, the percentage of engrafted cells that were (A) in cell-cycle phase S was determined via immunofluorescence analyses of Brdu incorporation, (B) proliferating was determined via immunofluorescence analyses of Ki67 expression, and (C) in cell-cycle phase M was determined via immunofluorescence analyses of PH3 expression. (D) Engrafted cells were identified via immunofluorescent staining for the expression of hcTnT, nuclei were counterstained with DAPI, and the percentage of engrafted cells that were undergoing apoptosis was determined via TUNEL staining. Images were from animals in the hiPSC-CCND2^OECM group. Bar=20 μm. *P<0.05 versus CCND2^WT. n=7 animals per treatment group at week 1; CCND2^WT (n=7) and CCND2^OE (n=11) at week 4; 4 sections per mice were evaluated.