Multimodal Spatial Transcriptomics Reveals the Developing Human Liver Niche at Single-Cell Resolution.

Background and Aims: A comprehensive understanding of the liver niche during development provides insights into a unique, naturally occurring multifunctional multicellular environment. We applied spatial transcriptomic and histologic approaches to identify and histologically validate RNA-based markers in human liver tissue during a critical developmental window when the liver serves as the primary site of hematopoiesis, just prior to the rapid growth phase of the third trimester. During this period, the developmental liver niche uniquely supports the coexistence of endodermal- and mesenchymal-derived cell populations that coordinate hematopoietic development and hepatocyte function. Methods: Nine formalin-fixed paraffin-embedded human developmental liver samples underwent histologic processing, staining, and evaluation prior to single-cell resolution spatial molecular imaging. Spatial transcriptomic profiling was followed by single-cell transcriptomic analysis and validation using multiplexed RNA fluorescent single-molecule in situ hybridization. Results: Single-cell spatial imaging identified epithelial, hematopoietic, endothelial, and stromal cell populations within shared microenvironments. This approach enabled molecular capture and histologic confirmation of 2 complex developmental phenomena: gene expression associated with the developing ductal plate and CXC motif chemokine ligand localization to the hepatic hematopoietic stem cell niche. Conclusion: Integration of markers identified through spatial transcriptomics will expand molecular panel design and enable more precise characterization of developmental biliary biology and pathobiology. Importantly, RNA-based understanding of the functional diversity and in situ spatial organization of the developmental liver niche will also be essential for authenticating and re-engineering these complex regenerative environments in vitro.