Thymic epithelial cell potency and fate in early organogenesis assessed by single cell transcriptional and functional analysis. [10x Genomics]

During development, cortical (c) and medullary (m) thymic epithelial cells (TEC) arise from the third pharyngeal pouch endoderm. Current models suggest that within the thymic primordium most TEC exist in a bipotent/common thymic epithelial progenitor cell (TEPC) state able to generate both cTEC and mTEC, at least until embryonic day 12.5 (E12.5) in the mouse. This view however, is challenged by recent transcriptomics and genetic evidence. We therefore set out to investigate the fate and potency of TEC in the early thymus. Here using single cell (sc) RNAseq we identify a candidate mTEC progenitor population at E12.5, consistent with recent reports. Via lineage-tracing we demonstrate this population is mTEC fate-restricted, validating our bioinformatics prediction. Using potency analyses we also establish that most E11.5 and E12.5 progenitor TEC are cTEC-fated. Finally we show that overnight culture causes most if not all E12.5 cTEC-fated TEPC to acquire functional bipotency, and provide a likely molecular mechanism for this changed differentiation potential. Collectively, our data overturn the widely held view that a common TEPC predominates in the E12.5 thymus, showing instead that sublineage-primed progenitors are present from the earliest stages of thymus organogenesis but that these early fetal TEPC exhibit cell-fate plasticity in response to extrinsic factors. Our data provide a significant advance in understanding of fetal thymic epithelial development and thus have implications for thymus-related clinical research, in particular research focussed on generating TEC from pluripotent stem cells. E12.5 and E13.5 wild type (WT), E12.5 and E13.5 Rosa26CreERt2;iFoxn1 (iFoxn1; called enforced Foxn1) and E12.5 and E13.5 Rosa26CreERt2 (called Cre-only) fetal thymi were microdissected. The samples processed for library preparation were as follows: (i) Freshly isolated wild type E12.5 and E13.5 and E12.5 iFoxn1 and Cre-only lobes were dissociated to single cell suspensions using TrypLE Express (Life Technologies 12604013) as above and immediately processed for library preparation; (ii) E12.5 wild type, iFoxn1 and Cre-only lobes were cultured overnight as intact lobes at the air-liquid interface (i.e. standard fetal thymic organ culture [FTOC] conditions, called FTOC), then processed to single cell suspensions using TrypLE Express; and (iii) E12.5 wild type, iFoxn1 and Cre-only lobes were dissociated to single cell suspensions and cultured overnight as monolayers (called monolayer), then harvested using TrypLE Express.