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

Abstract

ABSTRACTThe 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. Accordingly, regenerating thymic function for therapeutic purposes holds great 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 epithelial cell intrinsic and environmental signals that contribute to thymic morphogenesis is essential for our quest to derive thymic epithelial cells from pluripotent stem cells to reconstitute thymic function for clinical purposes.To deduce the signals instructing the development and specialization of the human thymic epithelial stroma, we have used a dual-trajectory single cell transcriptomic approach: (A) To delineate the trajectory from anterior foregut towards thymic fate and dissect alternate lineage bifurcations, 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 networks delineate cortical, and medullary thymic epithelial cells, conducting airway basal cells, respiratory bud-tip progenitor cells and esophageal basal cells. Activation of the NFKB/Interferon response axis, it the prevailing distinguishing feature that sets the thymic epithelium apart from all other anterior foregut derived organs. This correlates with highest interferon gamma protein expression by thymocytes in the thymic medulla. Unique expression patterns for the transcription factors SOX2 and TP63, and the relative expression of retinoic acid producing, and metabolizing enzymes are also observed. Within the thymic epithelial compartment, a cycling progenitor pool gives rise to three distinctive developmental trajectories. Cortical TECs and medullary TECs show distinctive lineage trajectories originating from a cluster of predominantly cycling TECs as early embryonic stages. Compared to medullary TECs, cortical TECs show increased Histone Deacetylase 4 expression paired with decreased levels of histone acetylation. Our studies trace thymus specific gene regulatory networks within the larger framework of epithelial cells from all major anterior foregut derived organs with the goal to provide insights into actionable pathways that help advance directed differentiation approaches for translational applications.ABSTRACT FIGURE