"INTRODUCTION: Myocardial infarction (MI) is an ischemic and inflammatory event majorly orchestrated by macrophages from infiltrating monocytes. These macrophages play a critical role in deciding the fate of the heart post-MI. However, there is no cardiac disease model in existence that incorporates an immune response. Hence, the aim of this project is to develop a humanized model of MI, using induced pluripotent stem cell (iPSC) derived cardiomyocytes together with inflammatory cytokine stimulation, to model the disease environment.
PURPOSE: Despite the advances in developing effective engineered heart tissue (EHT) models for MI that can recapitulate intricacies of the native myocardium, such as contractile properties and ability to respond to different chemical stimuli, there is still a need to make these models physiologically relevant. We hypothesize that the addition of macrophage-derived inflammatory cytokines can aid in making EHT models of MI more humanized.
METHODS: The first objective of this project is to obtain conditioned media obtained from immune cells for stimulating cardiomyocytes. In order to achieve this, iPSCs were differentiated to obtain macrophages (iMacs). Their expression of general macrophage (CD14, CD11b) and resident macrophage (CX3CR1, CCR2 and HLADR) markers were assessed, in addition to their phagocytic and polarization potentials. Next, iPSC-derived cardiomyocytes were obtained (iCMs) and the experssions of general cardiomyocyte (cTnT, cx43) and maturation (sarcomeric actinin) markers were assessed. Finally, polarizsation of iMacs were analyzed within a collagen/matrigel hydrogel system.
RESULTS: iMacs were found to be 93.6% CD14highCD11bhigh. Compared to blood-derived macrophages, CCR2 was downregulated and CX3CR1 and HLADR were upregulated in iMacs showing a resident macrophage phenotype. Additionally, iMacs also showed phagocytic potential (39.9%) and ability to be polarized to pro-inflammatory (on stimulation with LPS and IFN-gamma) and anti-inflammatory (on stimulation with IL4) states. iCMs were found to be positive for cTnT, cx43 and sarcomeric actinin. Additionally, they were found to be positive for MLC2a, denoting an atrial cardiomyocyte phenotype.
iMacs were found to not polarize within the collagen/matrigel hydrogel system, which will be used as the final model to develop the EHT.
CONCLUSIONS: Macrophages with a higher resident phenotype and have been generated. In the future, conditioned media from these iMacs stimulated with pro-inflammatory factors will be used to treat cardiomyocytes, to understand its effects on cardiomyocyte function."