Bone-derived stem cells repair the heart after myocardial infarction through transdifferentiation and paracrine signaling mechanisms

Abstract

Rationale: Autologous bone marrow-derived or cardiac-derived stem cell therapy for heart disease has demonstrated safety and efficacy in clinical trials, but functional improvements have been limited. Finding the optimal stem cell type best suited for cardiac regeneration is the key toward improving clinical outcomes.

Objective: To determine the mechanism by which novel bone-derived stem cells support the injured heart.

Methods and results: Cortical bone-derived stem cells (CBSCs) and cardiac-derived stem cells were isolated from enhanced green fluorescent protein (EGFP+) transgenic mice and were shown to express c-kit and Sca-1 as well as 8 paracrine factors involved in cardioprotection, angiogenesis, and stem cell function. Wild-type C57BL/6 mice underwent sham operation (n=21) or myocardial infarction with injection of CBSCs (n=67), cardiac-derived stem cells (n=36), or saline (n=60). Cardiac function was monitored using echocardiography. Only 2/8 paracrine factors were detected in EGFP+ CBSCs in vivo (basic fibroblast growth factor and vascular endothelial growth factor), and this expression was associated with increased neovascularization of the infarct border zone. CBSC therapy improved survival, cardiac function, regional strain, attenuated remodeling, and decreased infarct size relative to cardiac-derived stem cells- or saline-treated myocardial infarction controls. By 6 weeks, EGFP+ cardiomyocytes, vascular smooth muscle, and endothelial cells could be identified in CBSC-treated, but not in cardiac-derived stem cells-treated, animals. EGFP+ CBSC-derived isolated myocytes were smaller and more frequently mononucleated, but were functionally indistinguishable from EGFP- myocytes.

Conclusions: CBSCs improve survival, cardiac function, and attenuate remodeling through the following 2 mechanisms: (1) secretion of proangiogenic factors that stimulate endogenous neovascularization, and (2) differentiation into functional adult myocytes and vascular cells.

Keywords: differentiation; myocardial infarction; neovascularization; paracrine communication; stem cells.

Figure 1. In vitro characterization of stem cells

A) CBSC or CDC lysates were analyzed by Western analysis. Positive controls include mouse endothelial fibroblasts (MEF), liver, bone marrow (BM), and B lymphocytes (B Cell). Myocyte (MYO) lysates were used as negative controls for all samples. B) CBSCs (green) were fixed in vitro and immunostained against each paracrine factor (red). Nuclei are labeled with DAPI (blue) and scale bars = 20 μm. C) CBSCs or CDCs were allowed to proliferate over 72 hours and their culture media was analyzed by ELISA for the presence of soluble HGF, IGF, SCF, SDF-1, and VEGF. Samples were analyzed in triplicate and background signal was subtracted using unconditioned media blanks. NS = No Significant difference (p > 0.05).

Figure 2. Six-week survival

Mice underwent sham, MI+Saline, MI+CDC or MI+CBSC surgery. Data was analyzed using a Kaplan-Meier regression and significance was determined using the Log-Rank test.

Figure 3. Cardiac function measured by echocardiography

Animals underwent sham, MI+Saline, MI+CBSC or MI+CDC surgeries and received follow-up serial echocardiography at 1, 2, 4 and 6 weeks post-MI. A) Representative M-mode tracings from animals at baseline, 6 weeks post-MI+CBSC, 6 weeks post-MI+CDC, or 6 weeks post-MI+Saline. B) Structural and functional parameters derived from echocardiography measurements are shown. */**/***= CBSC v. Saline, */**/*** = CBSC v. CDC

Figure 4. Left ventricular endomyocardial strain

A) Three-dimensional regional wall velocity diagrams showing contraction (orange/positive values) or relaxation (blue/negative values) of 3 consecutive cardiac cycles. B) Vector diagrams showing the direction and magnitude of endocardial contraction at mid-systole. C) Global averages of strain and strain rate measured in the radial or longitudinal axes across the LV endocardium. */**/***= CBSC v. Saline, */**/*** = CBSC v. CDC

Figure 5. Characterization of paracrine factors secreted by stem cells after 24 hours post-MI in vivo

Animals receiving MI+CBSCs or MI+CDCs were sacrificed 24 hours post-MI for analysis of paracrine factor production. EGFP+ CBSCs stained positive for bFGF, and VEGF (shown in red), but negative for HGF, IGF-1, PDGF, SCF, and SDF-1. EGFP+ CDCs stained positive for Ang-1 in addition to bFGF and VEGF, but also stained negative for HGF, IGF-1, PDGF, SCF, and SDF-1. Nuclei are labeled with DAPI (blue) and injected CBSCs are green.

Figure 6. Expansion of EGFP+ CBSCs as stem cells engraft, align, and elongate over 6 weeks

Samples were immunostained against α-sarcomeric actin (red), nuclei are labeled with DAPI (blue) and injected EGPF+ CBSCs are green.

Figure 7. Cortical bone stem cells grow and differentiate over 6 weeks

A) 1 week post-MI and B) 2 weeks post-MI samples were immunostained for α-sarcomeric actin (red) and EGFP (green). 6 weeks post-MI samples were immunostained for C) α-sarcomeric actin (white), connexin43 (red) and EGFP (green) or D) α-smooth muscle actin (white), von Willebrand Factor (red) and EGFP (green). Nuclei were labeled with DAPI (blue) in all images.

Figure 8. Isolated myocyte size, number of nuclei, and cell physiology were analyzed 6 weeks post-MI+CBSC

A) Myocytes were immunostained against α-sarcomeric actin (red), EGFP (green), and DAPI (blue). B) Average surface area of EGFP+ versus EGFP- myocytes and percent of total EGFP+ or EGFP- cells that were mono-, bi-, or tetranucleated. C) Representative fractional shortening and Ca²⁺ transients of EGFP+ myocytes with 1 or 2 nuclei, or EGFP- myocytes with 1, 2, or 4 nuclei. Scale bars = 100 μm. D) Mean fractional shortening, peak Ca²⁺ F/F0, and the time constant of decay (τ) of Ca²⁺ transients for all EGFP+ versus EGFP- myocytes.