Poster U2 - Reconstruct native soluble cues in vitro to improve maturation of lung vascular models

Introduction: Diseases in pulmonary vasculature remain challenging to study due to the lack of functional ex vivo models. However, the cellular phenotypes and physiological functions in current vascular models poorly recapitulate the maturity and functionality of native vasculature, including lung vasculature, limiting their potential to model pathophysiology accurately. Paracrine signals are enriched in whole lung tissue and are critical in regulating endothelial maturation and vascular homeostasis. Here, we leveraged single-cell RNA-seq (scRNAseq) to identify cell-cell crosstalk signals in human lungs and determined that VEGF-D, Adrenomedullin, and Angiopoietin 1 could improve the maturation of primary human endothelial cells and iPSC-derived endothelial cells in 2D culture and in 3D bioreactor culture.

Materials and

Methods: Leveraging scRNAseq-based ligand-receptor Connectome analysis, we studied the cell-cell crosstalk signals in the human lung microvascular niche among healthy adult individuals. The impact of ligands on cellular phenotype and physiological functions was assessed in human lung microvascular endothelial cells (HLMVECs) and iPSC-derived endothelial cells (iPSC-ECFCs) in vitro 2D cell culture. The top screened ligands were further evaluated in the endothelial repopulated whole lung scaffolds.

Results, Conclusions, and Discussions: scRNAseq datasets reveal over 24 cell populations in the human lung microvascular niche. Within these cell populations, we identified top signal interactions between microvascular endothelium and surrounding cell populations, including SLIT2-ROBO4, ANGPT1-TEK, ADM-RAMP2, VEGFD-VEGFR2, BMP5-BMPR2, etc. Upon treatment with the top 10 identified ligands on both HLMVECs and iPSC-ECFCs in 2D culture, we found that VEGF-D, Adrenomedullin, and Angiopoietin 1 could improve vascular barrier function through Electrical Cell Impendence Sensing assay (ECIS) and transwell assay, and this effect is likely via actin cytoskeleton re-organization dependent mechanisms. We further determined that these ligands could suppress inflammatory signals by reducing ICAM1 and increasing I𝛋B𝛂 expression. In the endothelial repopulated lung vascular models, we determined the beneficial effect of the identified ligands on improving cellular phenotype toward native lung features and vascular barrier functions.
Taken together, this study highlights the cell-cell crosstalk ligand-receptor signals in the lung microvascular niche in healthy individuals and identifies potential native soluble ligands that may improve the cellular and physiological maturation of in vitro vascular models to further enhance fidelity for disease modelling applications.

Acknowledgements (Optional):