AIM  To investigate the source of “X particles” observed during the isolation of mouse bone marrow-derived macrophages (BMDMs) and to evaluate their effects on BMDM and myocardial cells.

METHODS  The size, morphology, and structure of “X particles” were analyzed using optical microscopy, scanning electron microscopy (SEM), and transmission electron microscopy (TEM). Quantitative real-time polymerase chain reaction (qRT-PCR) was employed to assess the expression of 16S and Rn18S genes within “X particles” and their influence on the expression of polarization-related genes in BMDMs (Cd80, Cd86, Il12, iNOS, Mrc1, Cd163, Arg1, Il10). RNA sequencing (RNA-seq) was performed to analyze the mRNA composition of “X particles” and their influence on the gene expression profile of BMDMs. The effect of “X particles” on the proliferation of HL-1 cardiomyocytes was evaluated using the CCK8 assay.

RESULTS  A significant number of “X particles” (diameter ≈5 μm) emerged on the third day of in vitro BMDM induction culture. These particles were categorized into two types based on morphology and content: enucleated red blood cells (minority) and lysosome-rich, anucleate cell-like structures (majority). “X particles” exhibited gene expression profiles most similar to those of monocytes, dendritic cells, and neutrophils among bone marrow-derived cells. Furthermore, “X particles” promoted BMDMs to maintain an unpolarized (M0) state (P<0.05) and significantly enhanced the proliferation of cardiomyocytes (P<0.01).

CONCLUSION  “X particles” are biologically active particles discovered during BMDM induction, characterized by strong refractive properties and a diameter of approximately 5 μm. They primarily consist of enucleated red blood cells and lysosome-rich cell-like structures. These particles likely originate from bone marrow-derived cells such as neutrophils, megakaryocytes, and monocytes. Importantly, “X particles” play a role in maintaining BMDMs in an unpolarized (M0) state and promote cardiomyocyte proliferation.