Primitive macrophages induce sarcomeric maturation and functional enhancement of developing human cardiac microtissues via efferocytic pathways.

Yolk sac macrophages are the first to seed the developing heart, however we have no understanding of their roles in human heart development and function due to a lack of accessible tissue. Here, we bridge this gap by differentiating human embryonic stem cells (hESCs) into primitive LYVE1+ macrophages (hESC-macrophages) that stably engraft within contractile cardiac microtissues composed of hESC-cardiomyocytes and fibroblasts. Engraftment induces a human fetal cardiac macrophage gene program enriched in efferocytic pathways. Functionally, hESC-macrophages trigger cardiomyocyte sarcomeric protein maturation, enhance contractile force and improve relaxation kinetics. Mechanistically, hESC-macrophages engage in phosphatidylserine dependent ingestion of apoptotic cardiomyocyte cargo, which reduces microtissue stress, leading hESC-cardiomyocytes to more closely resemble early human fetal ventricular cardiomyocytes, both transcriptionally and metabolically. Inhibiting hESC-macrophage efferocytosis impairs sarcomeric protein maturation and reduces cardiac microtissue function. Taken together, macrophage-engineered human cardiac microtissues represent a considerably improved model for human heart development, and reveal a major beneficial role for human primitive macrophages in enhancing early cardiac tissue function.

Human liver sinusoidal endothelial cells support the development of functional human pluripotent stem cell-derived Kupffer cells.

In mice, the first liver-resident macrophages, known as Kupffer cells (KCs), are thought to derive from yolk sac (YS) hematopoietic progenitors that are specified prior to the emergence of the hematopoietic stem cell (HSC). To investigate human KC development, we recapitulated YS-like hematopoiesis from human pluripotent stem cells (hPSCs) and transplanted derivative macrophage progenitors into NSG mice previously humanized with hPSC-liver sinusoidal endothelial cells (LSECs). We demonstrate that hPSC-LSECs facilitate stable hPSC-YS-macrophage engraftment for at least 7 weeks. Single-cell RNA sequencing (scRNA-seq) of engrafted YS-macrophages revealed a homogeneous MARCO-expressing KC gene signature and low expression of monocyte-like macrophage genes. In contrast, human cord blood (CB)-derived macrophage progenitors generated grafts that contain multiple hematopoietic lineages in addition to KCs. Functional analyses showed that the engrafted KCs actively perform phagocytosis and erythrophagocytosis in vivo. Taken together, these findings demonstrate that it is possible to generate human KCs from hPSC-derived, YS-like progenitors.

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

The intricate anatomical structure and high cellular density of the myocardium complicate the bioengineering of perfusable vascular networks within cardiac tissues. In vivo neonatal studies highlight the key role of resident cardiac macrophages in post-injury regeneration and angiogenesis. Here, we integrate human pluripotent stem-cell-derived primitive yolk-sac-like macrophages within vascularized heart-on-chip platforms. Macrophage incorporation profoundly impacted the functionality and perfusability of microvascularized cardiac tissues up to 2 weeks of culture. Macrophages mitigated tissue cytotoxicity and the release of cell-free mitochondrial DNA (mtDNA), while upregulating the secretion of pro-angiogenic, matrix remodeling, and cardioprotective cytokines. Bulk RNA sequencing (RNA-seq) revealed an upregulation of cardiac maturation and angiogenesis genes. Further, single-nuclei RNA sequencing (snRNA-seq) and secretome data suggest that macrophages may prime stromal cells for vascular development by inducing insulin like growth factor binding protein 7 (IGFBP7) and hepatocyte growth factor (HGF) expression. Our results underscore the vital role of primitive macrophages in the long-term vascularization of cardiac tissues, offering insights for therapy and advancing heart-on-a-chip technologies.

Trans-Activator Binding Site Context in RCNMV Modulates Subgenomic mRNA Transcription.

Many positive-sense RNA viruses transcribe subgenomic (sg) mRNAs during infections that template the translation of a subset of viral proteins. Red clover necrotic mosaic virus (RCNMV) expresses its capsid protein through the transcription of a sg mRNA from RNA1 genome segment. This transcription event is activated by an RNA structure formed by base pairing between a trans-activator (TA) in RNA2 and a trans-activator binding site (TABS) in RNA1. In this study, the impact of the structural context of the TABS in RNA1 on the TA-TABS interaction and sg mRNA transcription was investigated using in vitro and in vivo approaches. The results (i) generated RNA secondary structure models for the TA and TABS, (ii) revealed that the TABS is partially base paired with proximal upstream sequences, which limits TA access, (iii) demonstrated that the aforementioned intra-RNA1 base pairing involving the TABS modulates the TA-TABS interaction in vitro and sg mRNA levels during infections, and (iv) revealed that the TABS in RNA1 can be modified to mediate sg mRNA transcription in a TA-independent manner. These findings advance our understanding of transcriptional regulation in RCNMV and provide novel insights into the origin of the TA-TABS interaction.