Poster CC12 - Advanced engineered tissue model demonstrates principle of functional delegation

Introduction: Engineered tissue models enable the controlled evaluation of cellular community development and interactions in a highly biomimetic environment. We engineered whole lungs ex vivo to study the roles of cellular communities in tissue regeneration, self-organization, and the recapitulation of function. In particular, we leveraged single-cell RNA sequencing (scRNAseq) to tie patterns of cell phenotype and cell-cell signaling to tissue structure and function. In this way, we evaluated a primary principle of tissue biology, namely cellular delegation of non-cell-autonomous function (Okabe 2016). We observed gene expression patterns indicating functional delegation from client cells to accessory cells in the presence or absence of certain cell types. In this work, engineered tissues allowed us to validate biologic principles that are not possible to study in animal models.

Materials and

Methods: Native rat lungs were decellularized and utilized as structurally biomimetic, bioactive templates for tissue growth. Scaffolds were recellularized in a bioreactor by seeding endothelial cells into the vasculature, and epithelial, mesenchymal, and immune cells into the air compartment via the trachea. Experimental conditions included epithelial-only cultures (mono-culture), epithelium + endothelium (co-culture), epithelium + endothelium + mesenchyme (tri-culture), and epithelium + endothelium + mesenchyme + immune (quad-culture), which allowed us to study the effects of building multicellular communities on the tissue system. Following culture, engineered lungs were processed for single-cell RNA sequencing (scRNAseq) and analyzed using computational pipelines specially developed for engineered tissues. Cell-to-cell signaling was measured using NICHES (Raredon 2023) to evaluate ligand-receptor expression at the single-cell level.

Results, Conclusions, and Discussions: Histologically, the addition of alveolar macrophages to engineered lungs improved tissue architecture by reducing fibroblast overgrowth and reducing debris in air spaces (Fig. A-D). By NICHES analysis (Fig. E), we found that cell signaling shifted from tri- to quad-culture lungs, specifically regarding which cell type was supplying a particular ligand to another cell type. For example, in tri-culture lungs, epithelium was expressing Nrg4 to be received by Erbb2 also in epithelium (a pathway known to support epithelial survival and stem cell activation), but in quad-culture lungs, macrophages began supplying the ligand to epithelium instead (Fig. F). Another example is in tri-culture lungs, mesenchyme was expressing Lpl to be received by endothelium via Lrp2 (a pathway known to regulate lipoprotein metabolism) (Fig. G). However in quad-culture lungs, macrophages expressed this ligand to be received by endothelium instead. There were also several instances where alveolar macrophages in quad-culture lungs receive existing, “orphaned” signals through the expression of novel receptors. For example, receiving Cxcl6 signals from other cell types through the expression of Cxcr1 (a pro-inflammatory signal) (Fig. H). Several instances of these types of expression patterns appeared between culture conditions in pathways associated with growth factor signaling, cytokine signaling and metabolism.

In tri-culture lungs, there were several instances where other cell types expressed a needed ligand to a lesser degree than macrophages expressed in quad-culture lungs. This suggests instances of functional delegation, a hypothesized evolutionary phenomenon whereby “client” cells, such as epithelium or endothelium, achieve greater cell type specification by delegating certain necessary functions to “accessory” cells, such as mesenchyme or immune cells. Vestiges of these evolutionary patterns could be observed in this work when immune cells were not present to perform their typical support function and another cell type stepped in to perform the necessary function instead. The improved tissue organization in quad-culture lungs over tri-cultures could be due in part to the presence of required accessory cells serving the functions they had been delegated. The engineered lung system was an effective platform for validating this principle of functional delegation, which would not have been possible to study using animal models.

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