Visualizing the codevelopment of structural and immune cells in the human fetal lung using imaging mass cytometry

The codevelopment and interactions between structural and immune cells in the human fetal lung can be studied by using transcriptomic and proteomic approaches. Here, we used imaging mass cytometry with a 31-antibody panel to visualize immune and structural cell development in human fetal lung tissue from elective abortions across the pseudoglandular and canalicular stages, spanning postconception weeks (pcw) 6 (n = 1), 8 (n = 1), 10 (n = 1), 13 (n = 2), and 18 (n = 3). This approach allows us to map the developing structural components of the human fetal lung. During the early pseudoglandular stage, keratin-8 (KRT8⁺) epithelial structures appeared, gradually developing into KRT8⁺EpCAM⁺ budding tips and elongated luminal structures. By the late pseudoglandular and canalicular stages, these luminal structures were lined by KRT8⁺D2-40⁺ cells, with D2-40 (Podoplanin) known to be expressed in alveolar epithelial and lymphatic endothelial cells. Surrounding these structures were layers of α-smooth muscle actin⁺ cells. The immune compartment was predominantly myeloid in origin. CD206⁺CD68⁺ macrophages were present as early as the pseudoglandular stage, whereas HLA-DR⁺ myeloid cells appeared later around 13 pcw. Cellular interaction analysis revealed an accumulation of HLA-DR⁺ cells near the KRT8⁺EpCAM⁺ structural regions, suggestive of interactions between these cells during lung development. Our findings illustrate the dynamic development of structural and immune cell components in the human fetal lung throughout the pseudoglandular and canalicular stages. Furthermore, the observed interactions between structural and HLA-DR⁺ immune cells support the notion that immune-structural cross talk plays a role in human fetal lung development.NEW & NOTEWORTHY This study provides a detailed protocol to visualize the codevelopment of structural and immune cells in the human fetal lung through imaging mass cytometry (IMC). Using IMC, spatial relationships between lung epithelial cells and myeloid cells can be visualized, which may contribute to unraveling cellular interactions important for lung maturation.