A topographic atlas defines developmental origins of cell heterogeneity in the human embryonic lung [SC]. A topographic atlas defines developmental origins of cell heterogeneity in the human embryonic lung [SC]

The lung contains numerous specialized cell-types with distinct roles in tissue function and integrity. To clarify the origins and mechanisms generating cell heterogeneity, we created a comprehensive topographic atlas of early human lung development. Here, we report 83 cell states, several spatially-resolved developmental trajectories and predict cell interactions within defined tissue niches. We integrated scRNA-Seq and spatially resolved transcriptomics into a web-based, open platform for interactive exploration. We show distinct gene expression programs, accompanying sequential events of cell differentiation and maturation of the secretory and neuroendocrine cell-types in proximal epithelium. We define the origin of airway fibroblasts associated with airway smooth muscle in bronchovascular bundles and describe a trajectory of Schwann cell progenitors to intrinsic parasympathetic neurons controlling bronchoconstriction. Our atlas provides a rich resource for further research and a reference for defining deviations from homeostatic and repair mechanisms leading to pulmonary diseases. Overall design: For tissue dissociation, tracheas were removed and lungs were finely minced. For later timepoints, lobes were first dissected into smaller pieces. Then, they were digested in 4U/ml Elastase (Worthington, cat no. LS002292), 1 mg/ml of DNase (Worthington, cat no. LK003170) in HBSS (Gibco, cat no. 14170) at 37°C ranging between 30 min to 3 h depending on the age (older timepoints require longer digestion times). HBSS supplemented with 2 % FCS (Gibco, cat no. 10500064) was used for the whole procedure. The tissues were triturated with fire polished glass Pasteur pipettes every 15-20 min for the tissue to fall apart more easily and enhance the dissociation. After digestion, the cell suspension was filtered (twice if needed) in a 15ml Falcon tube using a 30μm cell strainer (CellTrics, Sysmex), to remove remaining undissociated clumps and debris. The filtered cell suspension was kept ice cold and was diluted (roughly 1:2) with ice cold HBSS to prevent further digestion by the enzyme.. The filtered cell suspension wascells were pelleted through centrifugation at 200g for 5 min at 4 °C, the supernatant was removed and the pellet resuspended in a small volume of calcium- and magnesium-free HBSS (Gibco, cat no.14170) and transferred to 1.5ml Eppendorf tubes precoated with 30% BSA (A9576, Sigma-Aldrich). A Bürker chamber was used for cell counting. NOTE FROM SUBMITTER: raw reads from our cohort are considered sensate and protected by GDPR regulations, and will therefore be uploaded to Swedish federated EGA.