A naturally derived cardiac extracellular matrix enhances cardiac progenitor cell behavior in vitro

Abstract

Myocardial infarction (MI) produces a collagen scar, altering the local microenvironment and impeding cardiac function. Cell therapy is a promising therapeutic option to replace the billions of myocytes lost following MI. Despite early successes, chronic function remains impaired and is likely a result of poor cellular retention, proliferation, and differentiation/maturation. While some efforts to deliver cells with scaffolds have attempted to address these shortcomings, they lack the natural cues required for optimal cell function. The goal of this study was to determine whether a naturally derived cardiac extracellular matrix (cECM) could enhance cardiac progenitor cell (CPC) function in vitro. CPCs were isolated via magnetic sorting of c-kit(+) cells and were grown on plates coated with either cECM or collagen I (Col). Our results show an increase in early cardiomyocyte markers on cECM compared with Col, as well as corresponding protein expression at a later time. CPCs show stronger serum-induced proliferation on cECM compared with Col, as well as increased resistance to apoptosis following serum starvation. Finally, a microfluidic adhesion assay demonstrated stronger adhesion of CPCs to cECM compared with Col. These data suggest that cECM may be optimal for CPC therapeutic delivery, as well as providing potential mechanisms to overcome the shortcomings of naked cell therapy.

Figure 1. Cardiogenic gene expression of CPCs cultured on cECM and COL

Cardiac progenitor cells (CPCs) were cultured on cECM (black bars) or COL (white bars) for 2 days and cardiomyocyte (A), fibroblast (B) and endothelial and smooth muscle (C) lineage markers evaluated by qPCR. Results were normalized to GAPDH and expressed as a fold change for cECM over COL (ΔΔCt) and reported as a mean ± SEM. Unpaired student’s t-test; *p<0.05, **p<0.01, n=4-6. COL = collagen, cECM = cardiac decellularized extracellular matrix, tnn = troponin, mhc = myosin heavy chain, fsp = fibroblast specific protein, vwf = von Willebrand factor, sm = smooth muscle, GAPDH = Glyceraldehyde-3-phosphate dehydrogenase.

Figure 2. Western analysis of cardiac protein expression

Protein was isolated from cardiac progenitor cells cultured on cECM (black bars) or COL (white bars) for 7 days. Grouped data (A) and representative blots (B) are shown as mean ± SEM. Images were quantified with ImageJ and protein expression was normalized to GAPDH. Unpaired student’s t-test; **p<0.01, ***p<0.001; n=4-6. COL = collagen, cECM = cardiac decellularized extracellular matrix, GAPDH = Glyceraldehyde-3-phosphate dehydrogenase.

Figure 3. Serum-induced proliferation of CPCs

Cardiac progenitor cells (CPCs) seeded on cECM (right) or COL (left) were cultured for 48 hours. Fold change in cell number was calculated as the final cell count divided by the number of cells seeded as determine by Coulter counting. Box and whisker plots show the mean, quartiles ± SEM. Paired student’s t-test, *p<0.05; n=7. COL = collagen, cECM = cardiac decellularized extracellular matrix.

Figure 4. Survival of serum-deprived CPCs cultured on cECM and COL

Cardiac progenitor cells (CPCs) cultured on cECM or COL were serum-deprived for 12 hours, then harvested for Annexin V staining. Representative histograms of Annexin V staining for CPCs cultured on cECM (black) and COL (blue) are shown (A). Gating was based on CPCs that were not serum-deprived (dotted line). Box and whisker plots (B) show mean, quartile ± SEM for grouped data. Paired student’s t-test; ***p<0.001; n=6. COL = collagen, cECM = cardiac decellularized extracellular matrix.

Figure 5. CPC adhesion to cECM and COL

Cardiac progenitor cell (CPC) adherence to cECM (black) and COL (blue) was determined by microfluidic adhesion assay where cells were subjected to increasing shear stresses. Grouped data shows mean ± SEM fraction of adherent cells (left-axis) over time with increasing shear stresses (dotted line, right-axis). CPC adherence to fibronectin and laminin were similar to COL (data not shown).

Figure 6. Extracellular matrix and adhesion molecule PCR array

Cardiac progenitor cells (CPCs) were cultured on either cECM (y-axis) or COL (x-axis) for 2 days and 3 samples from each condition were pooled for a total of 1 μg cDNA. PCR array plates were purchased from Qiagen (SABiosciences). Results are presented as logΔΔCt and considered significantly up- (red diamonds) or down- (green triangle) regulated for ± 2.5-fold changes for cECM compared to COL. Gray line represents no change in gene expression between conditions