Primitive macrophages promote a perivascular niche to enable vascular perfusion in human cardiac tissue
The intricate anatomical structure and high cellular density of the myocardium significantly complicate the bioengineering of vascular networks within cardiac tissues, creating challenges in establishing a perfusable and stable vasculature within these tissues. Emerging evidence from murine in vivo studies underscores the significant role of resident cardiac macrophages in facilitating cardiac regeneration post-injury, specifically their role in enhancing angiogenesis processes. Here, for the first time, we integrate human pluripotent stem cell derived macrophages, resembling primitive yolk-sac derived macrophages, within human in vitro vascularized heart-on-chip platforms.  The incorporation of primitive macrophages had a profound impact on the long term functionality of microvascularized cardiac tissue, particularly in enabling formation of perusable vasculature with a stable barrier function. These effects were contingent on physical cell-cell interactions and significantly diminished in transwell culture. The inclusion of primitive macrophages mitigated tissue cytotoxicity and curtailed the release of cell-free mitochondrial-DNA, underscoring their indispensable role for bioengineered human cardiac tissues. Furthermore, their incorporation upregulated the secretion of pro-angiogenic, matrix remodeling and cardioprotective cytokines such as MMP-12, MMP-2, Angiopoietin-like 1, NRG-3, SIGIRR and Adiponectin. RNA sequencing disclosed upregulation of cardiac maturation (TTNI3, SCN5A, MYL2,  and RYR2), and endothelial cells genes (PDGF-B, PECAM-1 and CDH5) indicating a decrease in endothelial cells death. Collectively, our results offer valuable insights into the integral role of primitive macrophages in directing long-term functional vascularization of cardiac tissues, paving the way for novel therapeutic strategies and advancing heart-on-a-chip systems. Overall design: RNA-seq was used to identify various effects in this study: 1. To investigate the effect of DPSCs, we performed RNA seq of fibrin tissues with only ECs vs. EC+DPSC 2. To investigate the effect of cardiomyocytes on vascularized tissues, we performed RNA seq of fibrin tissues with ECs+DPSC vs. EC+DPSC+CMs 2. To investigate the effect of macrophages on vascularized cardiac tissues, we performed RNA seq of fibrin tissues with ECs+DPSC+CMs vs. EC+DPSC+CMs+macrophages