Primitive macrophages enable long-term vascularization of human heart-on-a-chip platforms

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: To understand reciprocal interactions in our multicellular system, we performed single nucleus RNA sequencing (snRNA-seq) on three groups: 1) macrophage suspensions, 2) tissues comprising EC/DPSC/CM, and 3) tissues comprising of EC/DPSC/CM/MFs. Our overall goal was to assess how macrophages transcriptionally change due to other cells present in cardiac tissue, and further, to dissect intercellular communication and downstream pathways induced in ECs, cardiomyocytes and stromal cells driven by the presence of macrophages.心肌（myocardium）复杂的解剖结构与极高的细胞密度，极大地增加了心脏组织内血管网络生物工程构建的难度，使得在这类组织中建立可灌注且稳定的脉管系统面临诸多挑战。现有小鼠体内研究的新兴证据表明，驻留心脏巨噬细胞在损伤后心脏再生过程中发挥着重要作用，尤其是在促进血管生成（angiogenesis）方面。本研究首次将源自人多能干细胞（human pluripotent stem cell）、类似原始卵黄囊来源巨噬细胞的细胞，整合至人源体外血管化心脏芯片（heart-on-chip）平台中。原始巨噬细胞（primitive macrophages）的掺入对微血管化心脏组织的长期功能产生了显著影响，尤其助力形成了具备稳定屏障功能的可灌注脉管系统。这类效应依赖于细胞间的直接物理相互作用，在Transwell培养体系中其作用会显著减弱。原始巨噬细胞的加入减轻了组织细胞毒性，并减少了无细胞线粒体DNA（cell-free mitochondrial-DNA）的释放，这凸显了其对于生物工程化人源心脏组织不可或缺的作用。此外，巨噬细胞的掺入还上调了促血管生成、基质重塑与心脏保护类细胞因子的分泌，包括MMP-12、MMP-2、血管生成素样蛋白1（Angiopoietin-like 1）、NRG-3、SIGIRR以及脂联素（Adiponectin）。RNA测序（RNA sequencing）结果显示，心脏成熟相关基因（TTNI3、SCN5A、MYL2及RYR2）与内皮细胞相关基因（PDGF-B、PECAM-1及CDH5）均出现上调，表明内皮细胞的死亡有所减少。综上，本研究结果揭示了原始巨噬细胞在调控心脏组织长期功能性血管生成中的核心作用，为新型治疗策略的开发以及心脏芯片系统的优化提供了重要参考。整体实验设计：为解析本多细胞系统中的双向细胞互作，我们对三组样本开展了单细胞核RNA测序（single nucleus RNA sequencing, snRNA-seq）：1）巨噬细胞悬液样本；2）包含内皮细胞（Endothelial Cell, EC）/牙髓干细胞（Dental Pulp Stem Cell, DPSC）/心肌细胞（Cardiomyocyte, CM）的组织样本；3）包含EC/DPSC/CM/巨噬细胞（Macrophages, MFs）的组织样本。本研究的整体目标为：评估巨噬细胞因心脏组织中其他细胞的存在而发生的转录组变化，并进一步剖析由巨噬细胞介导的、在内皮细胞、心肌细胞与基质细胞间的细胞间通信及下游通路。