Single-cell RNA-Seq of Foxe1KO mouse ESC-derived cells

Functional thyroid follicles generation in vitro is obtained at high efficiency by doxycycline-mediated expression of Nkx2-1 and Pax8, two proteins involved in early specification of thyroid lineage. Although those factors are sufficient to induce thyroid specification in vitro, other players, such as Foxe1, are also present in early thyroid primordium and its loss-of-function is responsible for thyroid congenital defects in mice and humans. Here we show that Foxe1KO mESCs can be induced to differentiate towards thyroid lineage but the efficiency of thyroid follicular cells generated is drastically low. Moreover, correct expression of maturation genes and capacity to produce thyroid hormone in vitro are completely disrupted. On the other hand, Nkx2-1 expressing cells derived from Foxe1KO have an unexpected ability to deviate to a lung epithelium differentiation program and form organoids containing multiple lung cell types. These findings demonstrate that Foxe1 is required to reinforce thyroid cell fate in vitro and to promote terminal maturation of thyroid follicles. Overall design: At day22 of differentiation protocol, cell populations derived from Foxe1KO/Nkx2-1-T2a-mKO2 reporter mESC lines were isolated for scRNAseq profiling. GFP+, mKO2+ and mKO2- cells were sorted at different proportions (15%, 50%, 35%, respectively). Sorted cells were collected in PBS at a density of 800cells/ul and diluted accordingly to kit's instruction (10x Genomics Chromium Single Cell 3' v3). 12000 cells were loaded onto a channel of the Chromium Single Cell 3' microfluidic chip and barcoded with a 10X Chromium controller. Subsequently, RNA was reverse transcribed and amplified according to manufacturer's recommendations (10X Genomics). Library preparation (e.g. fragmentation, dA tailing, adapter ligation, indexing PCR) was performed based on 10x Genomics guidelines. Libraries were sequenced on a Illumina NovaSeq 6000 system.