Human cardiac progenitor cells engineered with Pim-I kinase enhance myocardial repair

Abstract

Objectives: The goal of this study was to demonstrate the enhancement of human cardiac progenitor cell (hCPC) reparative and regenerative potential by genetic modification for the treatment of myocardial infarction.

Background: Regenerative potential of stem cells to repair acute infarction is limited. Improved hCPC survival, proliferation, and differentiation into functional myocardium will increase efficacy and advance translational implementation of cardiac regeneration.

Methods: hCPCs isolated from the myocardium of heart failure patients undergoing left ventricular assist device implantation were engineered to express green fluorescent protein (hCPCe) or Pim-1-GFP (hCPCeP). Functional tests of hCPC regenerative potential were performed with immunocompromised mice by using intramyocardial adoptive transfer injection after infarction. Myocardial structure and function were monitored by echocardiographic and hemodynamic assessment for 20 weeks after delivery. hCPCe and hCPCeP expressing luciferase were observed by using bioluminescence imaging to noninvasively track persistence.

Results: hCPCeP exhibited augmentation of reparative potential relative to hCPCe control cells, as shown by significantly increased proliferation coupled with amelioration of infarction injury and increased hemodynamic performance at 20 weeks post-transplantation. Concurrent with enhanced cardiac structure and function, hCPCeP demonstrated increased cellular engraftment and differentiation with improved vasculature and reduced infarct size. Enhanced persistence of hCPCeP versus hCPCe was revealed by bioluminescence imaging at up to 8 weeks post-delivery.

Conclusions: Genetic engineering of hCPCs with Pim-1 enhanced repair of damaged myocardium. Ex vivo gene delivery to modify stem cells has emerged as a viable option addressing current limitations in the field. This study demonstrates that efficacy of hCPCs from the failing myocardium can be safely and significantly enhanced through expression of Pim-1 kinase, setting the stage for use of engineered cells in pre-clinical settings.

Figure 1. Enhancement of proliferation, mitochondrial activity and telomerase activity in hCPCeP

A) CyQuant assay: hCPCeP exhibit enhanced proliferation compared to hCPC and hCPCe for 3 days (n=4). B) CyQuant assay: hCPCeP treated with 10μM of quercetagetin show decreased proliferation relative to non-treated hCPCeP (n=4). C) Metabolic activity measurement by MTT reagent: hCPCeP demonstrate improved metabolic activity relative to hCPC and hCPCe (n=4). D) TERT activity is significantly higher in hCPCeP relative to hCPCe (n=3). E) Immunoblot analysis for p21 and p-p21 F) Quantitation of immunoblot (n=3). NS; non-significant, * p<0.05, **p<0.01, ***p<0.001

Figure 2. Increases in cardiac commitment of hCPCeP after dexamethasone differentiation

A) Quantitative RT-PCR analysis for hCPCe and hCPCeP after dexamethasone treatment for Mef2c, vWF and GATA-6 (n=3). *p<0.05, **p<0.01, ***p<0.001 hCPCeP-dex vs hCPCeP+dex; *p<0.05 hCPCe-dex vs hCPCe+dex; ^#p<0.05, ^##p<0.01, hCPCe+dex vs hCPCeP+dex,. B-C) Immunostaining for Mef2c (cardiac), vWF (endothelial), GATA-6 (smooth muscle) before and after dexamethasone treatment for 7 days.

Figure 3. Improvement of cardiac performance of mice treated with hCPCeP 20 weeks after transplantation in SCID mice

A-B) Percent of fractional shortening (FS) and ejection fraction (EF) measured by echocardiography, sham n=6, vehicle n=12, hCPCe n=20, hCPCeP n=20. C) Hemodynamic assessment dp/dt after cell transplantation (sham n=3, vehicle n=6, hCPCe n=6, hCPCeP n=6). D) Left Ventricular Developed Pressure (LVDP). *p<0.05, **p<0.01, ***p<0.001 for vehicle vs hCPCe, φp<0.05, φφp<0.01, φφφp<0.001 for vehicle vs hCPCeP, #p<0.05, ##p<0.01, ###p<0.001 for hCPCe vs hCPCeP.

Figure 4. hCPCeP show increases in telomere length, enhancement of c-kit positive cell number and decreases in fibrosis

A) remote and B) infarct zone telomere length in mice treated with hCPCeP, telomere (white), GFP (green), desmin (red), nuclei (blue). C) Quantitation of telomere length of hCPCe and hCPCeP (p<0.05). D) Masson’s Trichrome staining for vehicle, hCPCe and hCPCeP injected SCID mice. E) Percentage of infarcted left ventricular free wall (LVFW) in vehicle, hCPCe and hCPCeP (n=3) p<0.01. F-G) Immunostaining for c-kit (white), GFP (green), α-sarcomeric actin (red) and nuclei 24 (blue) in hCPCe and hCPCeP respectively. H) Quantitation of total number of c-kit+ cells/mm2 in vehicle, hCPCe and hCPCeP. I) Quantitation of GFP+ and c-kit+ cells/mm² in hCPCe and hCPCeP treated animals (n=3).

Figure 5. hCPCeP augment myocardial repair 12 weeks after transplantation

Immunolabeling for A) α-sarcomeric actin (red), GFP (green) and nuclei (blue). B) SM22 (white), GFP (green), α-sarcomeric actin (red) and nuclei (blue). C) vW (white), GFP (green), α-sarcomeric actin (red) and nuclei (blue). D) Quantitation of SM22, vWF and α-actin in hCPCe and hCPCeP (p<0.001). Boxes indicate enlarged areas. Scale bars = 150μm for all panels except row A, where hCPCe and hCPCeP widefield images = 50μm and insets – 25μm.

Figure 6. Enhancement of hCPCeP persistence

A) Firefly luciferase reporter construct B) BLI imaging: Pseudocolor images representing signal intensity in mice treated with hCPCe-Luc (top) and hCPCeP-Luc (bottom) after myocardial infarction. hCPCe-Luc signal is not detected 7 days after transplantation while hCPCeP-luc shows signal throughout the experimental cohort for 56 days C) Quantitation of pseudocolor images represented in maximum radiance (p/s/cm²/sr).

Figure 7. Echocardiography and MRI of mice 8 weeks after Myocardial infarction

A) echocardiographic images from hCPCe -Luc and hCPCeP-Luc, B) LVEDd, C) LVESd, D) Anterior wall thickness (AW). (E-H) MRI: E) MRI images, F) LVEDV, G) Ejection fraction %, H) LVESV. *p<0.05 vs hCPCe