Combinatorial Polymer Electrospun Matrices Promote Physiologically-Relevant Cardiomyogenic Stem Cell Differentiation

Myocardial infarction results in extensive cardiomyocyte death which can lead to fatal arrhythmias or congestive heart failure. Delivery of stem cells to repopulate damaged cardiac tissue may be an attractive and innovative solution for repairing the damaged heart. Instructive polymer scaffolds with a wide range of properties have been used extensively to direct the differentiation of stem cells. In this study, we have optimized the chemical and mechanical properties of an electrospun polymer mesh for directed differentiation of embryonic stem cells (ESCs) towards a cardiomyogenic lineage. A combinatorial polymer library was prepared by copolymerizing three distinct subunits at varying molar ratios to tune the physicochemical properties of the resulting polymer: hydrophilic polyethylene glycol (PEG), hydrophobic poly(ε-caprolactone) (PCL), and negatively-charged, carboxylated PCL (CPCL). Murine ESCs were cultured on electrospun polymeric scaffolds and their differentiation to cardiomyocytes was assessed through measurements of viability, intracellular reactive oxygen species (ROS), α-myosin heavy chain expression (α-MHC), and intracellular Ca2+ signaling dynamics. Interestingly, ESCs on the most compliant substrate, 4%PEG-86%PCL-10%CPCL, exhibited the highest α-MHC expression as well as the most mature Ca2+ signaling dynamics. To investigate the role of scaffold modulus in ESC differentiation, the scaffold fiber density was reduced by altering the electrospinning parameters. The reduced modulus was found to enhance α-MHC gene expression, and promote maturation of myocyte Ca2+ handling. These data indicate that ESC-derived cardiomyocyte differentiation and maturation can be promoted by tuning the mechanical and chemical properties of polymer scaffold via copolymerization and electrospinning techniques

EB differentiation on polymer fiber scaffolds in the presence of DMH1.

<p>EBs with and without DMH1 treatment were stained for SERCA2a, an indicator of active Ca²⁺ transient. EBs on 4%PEG-86%PCL-10%CPCL exhibited enhanced expression of SERCA2a, compared to untreated EBs on PCL scaffolds or glass alone. Scale bars = 50 µm.</p

EB attachment and protein expression.

<p>(<b>a</b>) Phase contrast and fluorescence images of EBs are shown at day 10. Cells were cultured on gelatin coated glass cover slips with and without 4%PEG-86%PCL-10%CPCL copolymer scaffolds. Cells grown on 4%PEG-86%PCL-10%CPCL maintained more adhered, circular EBs and (<b>b</b>) exhibited higher α-MHC protein expression. Scale bars = 10 µm. * <i>p</i><0.0005 versus control.</p

Schematic representation of polymer synthesis PEG-PCL block copolymers were first synthesized by ring-opening polymerization.

<p>CPCL was then randomly added within the PCL subunit. Polymers are abbreviated <i>x</i>%PEG-<i>y</i>%PCL-<i>z</i>%CPCL.</p

Characterization of polymer properties.

<p>M_n was measured by GPC according to dn/dc light scattering values. Wet and dry moduli were calculated from stress/strain measurements.</p>a<p>Molecular weight measured by GPC in THF,</p>b<p>Measured on a uniaxial Bose ElectroForce 3100 mechanical tester,</p>c<p>Measured by DMA.</p