applications such as drug screening, proteomics, or Hi-C analysis. To overcome these limitations, we previously established immortalized erythroblast and megakaryocyte progenitor lines using defined factors, enabling long-term expansion and efficient platelet production 7–9). Building on this, we developed a doxycycline (Dox)-inducible immortalization system for generating immortalized macrophages (imMφ), vascular endothelial cells (imVECs), and smooth muscle cells (imVSMCs) from human iPSCs (Paul et al., unpublished; Japanese Patent: 2021-109513) (Fig. 4A). These cells can be expanded long-term and matured upon Dox withdrawal (Fig. 4B). RNA-seq analysis revealed that imVECs form three distinct clusters in principal component analysis (PCA) and gene expression profile: iPSCs, Dox-ON (immature), and Dox-OFF (mature), with the Dox-OFF cells closely resembling primary endothelial cells (Fig. 4C and D). These results suggest that our immortalized cell platform enables scalable and functionally relevant cell production for diverse biomedical applications.Figure 4. lmmortalization and characterization of iPS-derived cells.Using these immortalized cells, we established a 2D co-culture system that recapitulates early atherosclerotic changes. Matured imMφ were stimulated with oxidized low-density lipoprotein (oxLDL), lipopolysaccharide (LPS), or both for 6 hours, then co-cultured with matured imVECs and imVSMCs after thorough washing. After 72 hours, CD11b¯ imVECs and imVSMCs were isolated by cell sorting for further analysis. LPS-treated imMφ notably increased ICAM1 mRNA and surface expression in imVECs, while contractile markers (ACTA2, CNN1) were reduced in imVSMCs, indicating phenotypic switching to a synthetic state (Fig. 5A and B) (Liu et al., unpublished).To assess the model’s utility for drug screening, we treated the co-cultures with rosuvastatin and reparixin. Rosuvastatin reduced ICAM1 expression in imVECs compared to the oxLDL+LPS group. Reparixin enhanced contractile marker expression in imVSMCs, suggesting a protective effect (Fig. 6A and B) (Liu et al., unpublished). These findings demonstrate the model’s potential for studying early atherosclerosis and evaluating therapeutic interventions. ― 247 ―
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