Layered smooth muscle cell-endothelial progenitor cell sheets derived from the bone marrow augment postinfarction ventricular function

Abstract

Objective: The angiogenic potential of endothelial progenitor cells (EPCs) may be limited by the absence of their natural biologic foundation, namely smooth muscle pericytes. We hypothesized that joint delivery of EPCs and smooth muscle cells (SMCs) in a novel, totally bone marrow-derived cell sheet will mimic the native architecture of a mature blood vessel and act as an angiogenic construct to limit post infarction ventricular remodeling.

Methods: Primary EPCs and mesenchymal stem cells were isolated from bone marrow of Wistar rats. Mesenchymal stem cells were transdifferentiated into SMCs by culture on fibronectin-coated culture dishes. Confluent SMCs topped with confluent EPCs were detached from an Upcell dish to create a SMC-EPC bi-level cell sheet. A rodent model of ischemic cardiomyopathy was then created by ligating the left anterior descending artery. Rats were randomized into 3 groups: cell sheet transplantation (n = 9), no treatment (n = 12), or sham surgery control (n = 7).

Results: Four weeks postinfarction, mature vessel density tended to increase in cell sheet-treated animals compared with controls. Cell sheet therapy significantly attenuated the extent of cardiac fibrosis compared with that of the untreated group (untreated vs cell sheet, 198 degrees [interquartile range (IQR), 151-246 degrees] vs 103 degrees [IQR, 92-113 degrees], P = .04). Furthermore, EPC-SMC cell sheet transplantation attenuated myocardial dysfunction, as evidenced by an increase in left ventricular ejection fraction (untreated vs cell sheet vs sham, 33.5% [IQR, 27.8%-35.7%] vs 45.9% [IQR, 43.6%-48.4%] vs 59.3% [IQR, 58.8%-63.5%], P = .001) and decreases in left ventricular dimensions.

Conclusions: The bone marrow-derived, spatially arranged SMC-EPC bi-level cell sheet is a novel, multilineage cellular therapy obtained from a translationally practical source. Interactions between SMCs and EPCs augment mature neovascularization, limit adverse remodeling, and improve ventricular function after myocardial infarction.

Keywords: cell sheet; myocardial infarction; neovascularization; regeneration; stem cells; tissue engineering.

Figure 1

Nanovolume capillary electrophoresis-based protein analysis was performed on cultured mesenchymal stem cells (MSCs) and transdifferentiated smooth muscle cells (SMCs) (MSC, n=3; SMC, n=3). (A) Electropherogram and system software generated peak area and molecular weight data linear analysis for alpha smooth muscle actin (SMA), SM22 alpha, and caldesmon between MSCs and SMCs. Green indicates SMC; blue, MSC. (B) Representative immunoblots for alpha SMA, SM22 alpha, and caldesmon between MSC and SMC. (C) Transdifferentiated SMCs demonstrated higher protein concentrations of alpha SMA, SM22 alpha, and caldesmon than MSCs. The intensity level of detected protein bands was standardized by dividing by the intensity level of vinculin.

Figure 2

(A) The bi-level cell sheet maintained alpha smooth muscle actin (SMA) positive smooth muscle cells (SMCs) and CD31 positive endothelial progenitor cells (EPCs) in separate layers in vitro. Green indicates alpha SMA; red, CD31. White bar = 100 um. (B) Representative images demonstrating von Willebrand factor (vWF) and alpha smooth muscle actin (SMA) staining of border zone myocardium for cell sheet-treated and untreated groups. Green indicates alpha SMA; red, vWF. (C) Quantification of arterial density. Mature artery density was increased in the cell sheet-treated group compared with the untreated group.

Figure 3

(A) Representative Masson’s trichrome staining of the heart for cell sheet-treated (n=4), untreated (n=4), and sham control (n=4) groups. (B) Quantification of cardiac fibrotic extension. Fibrosis was significantly suppressed in the cell-sheet group compared with the untreated group.

Figure 4

(A) Representative cardiac magnetic resonance images at end-diastolic (ED) and end-systolic (ES) phases for cell sheet-treated (n=9), untreated (n=12), and sham control (n=7) groups. Examinations were performed 4 weeks after myocardial infarction. (B) Remarkable differences were noted between the three groups with respect to left ventricular end-diastolic volume (LVEDV), left ventricular end-systolic volume (LVESV), and ejection fraction (LVEF).