Figure 2
Foxn1 gene transduction and phenotypic/functional characterization of iPSC-TECs. (a) TEC differentiation with Foxn1-expressing mouse iPSC. Fluorescent images show double staining for Foxn1 (red) and the nucleus (blue). Scale bars represent 100 μm. Flow cytometric plots show representative flow cytometric analysis of TEC-related marker molecules on day 14 of differentiation. (b) Fold induction efficiency of marker-expressing cells compared with normal (not transduced with Foxn1) iPSCs. (c) Expression of Hoxa3, Tbx1, Pax9, Pax1, Ccl25, Dll4, and Foxn1-UTR on day 14 of differentiation. Foxn1-UTR expression was analysed by primers specific for 3′-UTR regions of Foxn1 mRNA. (n = 8, biological replicates). (d) Expression of “Total” and “Endogenous” Foxn1 on day 14 of differentiation (n = 4, biological replicates). (e) Schematic of Foxn1-iPSC-derived transplantation of TEC into nude recipients (left). iPSC-TECs (EpCAM+, Ly51+, UEA-1+) were sorted, and aggregates (1 × 105 iPSC-TECs were mixed with 3 × 104 MEFs) were transplanted into nude mice. Peripheral blood analysis of nude mice 6 weeks after transplantation. Nude mice received iPSC-TEC aggregates without DN1 thymocytes. Plots show live cell (DAPI−) and recipient blood cell (CD45.2+) gated populations (right). (*p < 0.05, **p < 0.01, Student’s t-test).
