Abstract
Cardiovascular disease (CVD) remains the leading cause of death in the United States with a range of treatments that vary according to the function that is comprised and patient-case severity. Despite progress in medicine and biomedical research, current cellular therapies are incapable of repairing and restoring cardiac function for heart-related CVDs that stem from dysfunctional cardiomyocytes (CM) or cell death. Since the human heart is incapable of regenerating itself naturally, a possible therapeutic strategy is to use human induced pluripotent stem cells (hiPSCs) to derive autologous CMs for replacing nonfunctioning or diseased cells. However, producing sufficient quantities of functionally suitable contractile and pacemaking CM subtypes poses a fundamental hurdle. Cellular interactions with the extracellular matrix (ECM) have been shown to transduce critical signals for cell-lineage specification. Previous studies that investigated the interactions between hiPSC-derived CMs and ECM proteins have shown that protein composition provides biochemical cues that are responsible for phenotypic maintenance and development. Additionally, prior studies that examined the interplay between human pluripotent stem cells (iPSC and ESC) and ECM elasticity have demonstrated defined substrate stiffness induces stem cell differentiation and lineage specification. In addition, these studies have indicated ECM stiffness provides biomechanical cues for CM functional maturation. HiPSC directed cardiogenesis protocols have improved since their inception, but generating pure and functionally mature populations of hiPSC-derived CMs remains a prominent issue. Based on these findings, the ECM has a necessary presence that is absent in feeder-free hiPSC-derived CM cultures. The primary goal of the Lieu laboratory is to investigate the differentiation, enrichment and maturation of hiPSC-derived derived pacemaking and contractile CMs. As a way to contribute to this goal, we examined how the ECM influences CM subtype specification and phenotype maintenance by evaluating properties of the ECM independently to determine the mechanisms by which the ECM niche facilitates differentiation and CM lineage specification into pacemaking and contractile subtypes. We hypothesized that the biochemical and biomechanical properties of the ECM could promote CM subtype specification and facilitate individual functional phenotype maintenance. Our study was organized in two specific aims. The first aim was to determine the reprogramming effects of the ECM microenvironment on hiPSC-derived CM subtype plasticity by performing immunocytochemical (ICC) staining of hiPSC-derived CM markers to quantify protein expression and optical recording of hiPSC-derived action potentials in vitro. The second aim was to determine the effects of the ECM on hiPSC-derived cardiac progenitor cell (CPC) differentiation into contractile and pacemaking CM subtypes by performing immunocytochemical (ICC) staining of hiPSC-derived CM markers in vitro to quantify protein expression. Here, we demonstrated that the expression of pacemaking, contractile, and integrin-binding markers were dependent on different variables of the ECM during hiPSC-derived CM reprogramming and hiPSC-derived CPC differentiation. Furthermore, the electrophysiological properties and subtype distribution of hiPSC-derived CMs were dependent on the unique combination of ECM protein coating and elasticity of the ECM.