Single cell transcriptomics of human prenatal anterior foregut-derived organs identifies distinct developmental signatures directing commitment and specialization of the thymic epithelial stroma

The thymus is the primary lymphoid organ that instructs adaptive T cell immunity and central tolerance. Within the thymus, the thymic epithelium comprises a diverse and highly specialized set of cells that promote T cell maturation, proliferation, and selection of a diverse and self-tolerant T cell receptor repertoire. As such, the function of the thymic epithelium is central to the integrity of the immune system in health and disease and regenerating thymic function holds great therapeutic promise. However, the gene regulatory networks that drive thymic epithelial cell ontogeny and maintenance during human embryonic development remain incompletely understood. Elucidating the complex interplay between cell intrinsic and environmental signals that contribute to thymic morphogenesis is essential to derive thymic epithelial cells from pluripotent stem cells to reconstitute thymic function for therapeutic purposes. To deduce the signals instructing the development and specialization of the human thymic epithelial stroma, we have used a dual single cell transcriptomic approach: (A) To dissect lineage bifurcations on the trajectory towards thymic fate, we have compared transcriptional signatures of epithelial cells from human fetal thymus to epithelial cells derived from the other major anterior foregut-derived organs, i.e., bronchus, lung, and esophagus as well as the parathyroid. (B) To define the proliferative dynamics that give rise to developmental hierarchies within specialized thymic epithelial compartments, we have compared human thymus samples sequentially during embryonic, fetal, and early postnatal stages of organogenesis. Distinct gene regulatory patterns delineate cortical, and medullary thymic epithelial cells, conducting airway basal cells, respiratory bud-tip progenitor cells and esophageal basal cells. Specifically, the transcription factors SOX2 and TP63, that promote basal epithelial cell fate when co-expressed and drive branching morphogenesis when cycled in a coordinated fashion, show a uniquely divergent TP63highSOX2low expression pattern in the thymic epithelium. In addition, the thymic epithelium shows distinctive interferon and NFkB-driven signature which is not present in other epithelial cells. Within the thymic epithelial compartments, a cycling progenitor pool gives rise to three distinct developmental trajectories. A dichotomy between PI3Kinase and TGFb signaling between cTECs and mTECs was observed. Our studies map gene regulatory signatures during thymic epithelial cell ontogeny with the intent to advance directed differentiation approaches for translational applications. ABSTRACT FIGURE