Engineering Combinatorial Microenvironments for Structural and Functional Maturation of Human Stem Cell-Derived Cardiomyocytes

Abstract

As cardiovascular disease remains to be the leading cause of death worldwide, cardiac regenerative medicine aims to apply design methods to develop functional cardiac tissue for directed therapy as well as <italic>in vitro</italic> screening assays. Research in this area has shown varying degrees of success, but fully functional cardiac tissue remains to be achieved. This short–coming is due to failures in mimicking native heart tissue in vitro. The extracellular matrix (ECM) of the heart is a complex structure responsible for both biochemical and mechanical cues to the surrounding myocardium. Past research has relied heavily on the use of native biochemical signals of the ECM to influence cardiomyocyte function, but the mechanical signals of heart ECM have been less studied. The ECM of the heart is made up of aligned collagen fibers as well as other important proteins in the basement membrane responsible for cell–cell and cell–ECM interactions. The nanoscale collagen fibers have been shown to play a major role in the structural architecture of the overlying macroscopic myocardium. Advancements in nanofabrication techniques have made it possible to study the effect of substrate nanotopography on cardiomyocyte structure and function. The proteins of the basement membrane including laminin and fibronectin have been shown to strongly influence the adhesion of cardiomyocytes through integrin interactions. Recently, a specific repeating amino acid sequence, Arg–Gly–Asp (RGD), found in many native adhesion proteins, has been shown to promote cell adhesion in vitro<super>1,2</super>. Here we present a platform in which we are able to study the effect of nanoscale structural cues as well as ECM biochemical signals on maturation of human pluripotent stem cell–derived cardiomyocytes (hPSC–CMs). Using a customized 4 x 4 island nanopatterned substrate, nanogroove widths ranging from 350nm to 2000nm were investigated. We also present the synthesis and incorporation of bifunctionalized peptide, PUA binding peptide–RGD (PUABP–RGD) into the platform to further study the effect of native ECM–like biochemical cues on the structural maturation of hPSC–CMs.