Conditioning thymic epithelial cell differentiation

The thymus is of endodermal origin, sharing its ancestor with respiratory or gastric organs such as the lung, liver, or pancreas13. We first focused on constructing a step-by-step protocol for induction of TECs through definitive endoderm (DE), anterior foregut endoderm (AFE), and pharyngeal endoderm (PE) (Fig. 1a). DE is known to be induced by a high concentration of Activin and is defined by cell surface expression of Cxcr4, c-Kit, and EpCAM14. We established a protocol for DE induction by modifying several induction methods to optimize them for our iPS cell line (Supplementary Figs. 1–4). Flow cytometric analysis revealed highly overlapped expression of these marker molecules, suggesting efficient (c-Kit+Cxcr4+ cells were 86.7% ± 3.25) DE induction (Fig. 1b). Additionally, upregulation of DE marker genes, defined by quantitative PCR (qPCR), also indicated appropriate differentiation (Fig. 1c). We also examined Foxa2 protein expression and found it to be primarily localized to the nucleus, whereas protein expression of Sox2, one of the key factors responsible for pluripotency, was not detected (Supplementary Fig. 5). These results are consistent with the estimated efficiency determined by flow cytometry (Fig. 1b).

Figure 1 Generation of iPSC-TECs. (a) Schematic showing the differentiation protocol of thymic epithelial cells. (b) Definitive endoderm marker expression on day 5 of differentiation. Plots show representative flow cytometric analysis. (c) Expression of Foxa2, Sox17, Cer1, and Oct4 on day 5 of differentiation. Definitive endoderm markers (biological replicates: Foxa2, n = 8; Sox17, n = 7; Cer1, n = 6) and Pluripotency gene (Oct4, n = 8). (d) RT-qPCR analysis of anterior foregut marker gene expression on day 7 of differentiation. Definitive endoderm cells were treated with SB431542 and LDN193189 for 2 days (n = 3, biological replicates). (e) Double immunostaining for Foxa2 (green) and Sox2 (red). Nuclei were counterstained with DAPI (blue). Lower panels show enlargement of insets in upper panels. Scale bars represent 200 μm. (f) RT-qPCR analysis of Tbx1 and Hoxa3 (n = 6 and n = 7, respectively, biological replicates) as pharyngeal pouch endoderm marker genes on day 9. (g) Optimizing pharyngeal endoderm differentiation conditions. Each bar represents culture condition without the indicated factors. NS, not significant, Tukey’s multiple comparison test (n = 3, biological replicates). (h) RT-qPCR analysis for TEC-related genes (Hoxa3, Foxn1, and Dll4) on day 14 of differentiation (n = 3, biological replicates). (i) Representative plots of cell surface analysis of TEC-related marker molecules and average induction rate of each marker-positive cell (n = 3, biological replicates). Plots show live cell (DAPI−) gated population. *p < 0.05, **p < 0.01, two-tailed Student’s t-test. DE: definitive endoderm; AFE: anterior foregut endoderm; PE: pharyngeal endoderm; TEC: thymic epithelial cell. Full size image

The thymus arises from an anterior portion of developing endoderm, AFE13. The early anterior-posterior formation is regulated by bone morphogenetic protein (BMP), fibroblast growth factor (FGF), and Wnt/β-catenin signalling. Previously, screening multiple combinations of signal agonists and antagonists revealed that DE cells could be anteriorized by simultaneous inhibition of transforming growth factor (TGF) and BMP signaling15. By culturing DE cells in the presence of these signal inhibitors, i.e., SB431542 and LDN193189, anterior marker genes were significantly upregulated, and immunofluorescence imaging showed merged expression of Sox2 with Foxa2 (Fig. 1d,e). Importantly, suppressed Sox2 expression at the DE stage was restored after the anteriorization process. To induce pharyngeal endoderm, anterior foregut endoderm cells were exposed to 6 factors to stimulate BMP, FGF, Wnt/β-catenin, and retinoic acid (RA) signalling whilst under combinatory inhibition of TGF and the sonic hedgehog signalling pathway10,15. This induction step efficiently induced pharyngeal pouch marker Tbx1 and Hoxa3 (Fig. 1f). Because TECs are known to develop from the Hoxa3-expressing region in the 3rd pharyngeal pouch (3rd p.p.)16,17, we focused on optimizing culture conditions by using Hoxa3 as a guide for sufficient 3rd p.p. induction. We identified that single or combinatory withdrawal of BMP4, CHIR99021, or cyclopamine from the induction conditions did not affect Hoxa3 and Tbx1 expression on day 9 (Fig. 1g). However, subtracting FGF8, SB431542, or RA resulted in reduced expression of Tbx1 and Hoxa3 (data not shown).

We found that thymic specification can be carried out by continuous exposure to FGF8, SB431542, and RA until day 14 of differentiation (morphological changes are shown in Supplementary Fig. 5). Physiological TEC differentiation is dependent on region-specific expression of forkhead box protein, Foxn118. Hence, we employed Foxn1 as a key differentiation marker and quantified its expression on day 14. A small but significant increase in Foxn1 expression indicated the existence of a TEC population in the obtained cells (Fig. 1h). We also analysed marker molecules on the cell surface by flow cytometry. TECs are categorized as an epithelial cell subset, and approximately 5% of obtained cells expressed epithelial cell adhesion molecule (EpCAM). TECs are further classified into two subsets based on their surface expression of Ly51 and UEA-1, which are expressed by cortical and medullary TECs, respectively19. Flow cytometry analysis revealed minimal levels of these populations (Fig. 1i). Bilateral cross-talk between developing thymocytes and TEC progenitors is an essential event during mature TEC development1. However, it is indicated by cell surface analysis that the in vitro culture condition promoted terminal differentiation without receiving signals from T cells. Notably, gene expression of delta-like 4 ligand (Dll4) was also determined by qPCR, and the results suggest the possibility of T-cell differentiation (Fig. 1h).

Transduction of exogenous Foxn1 gene promotes iPSC-TEC differentiation

Foxn1 is known as a key transcription factor of thymus morphogenesis. Loss-of-function mutation of Foxn1 results in athymia and nude phenotype because of defects in hair follicle formation20. We next sought to address whether this developmental control gene would also affect in vitro generation of TECs. To evaluate this possibility, we generated a mouse iPS cell line carrying an exogenous gene construct that encodes mouse Foxn1 under the human EF1α promoter. We did not observe any change in cell growth activity or toxicity with Foxn1-transduced iPSCs (data not shown). TEC induction was carried out by following our induction protocol (Figs. 1a, 2a). Flow cytometric analysis revealed that inducing Foxn1 expression during in vitro differentiation had a promotive effect on TEC induction (Fig. 2b). Comparing the differentiation results showed the significantly enhanced induction efficiency of TEC-related molecule expressing cells (Fig. 2b).

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). Full size image

We also analysed developmental genes such as Hoxa3, Tbx1, and Pax9, which showed a significant increase through differentiation induction (Fig. 2c). Additionally, Ccl25 and Dll4 were also upregulated (Fig. 2c). As it is known that the molecular expression of Ccl25 and Dll4 can be influenced by, but not dependent on21,22,23, Foxn1, there was a possibility that the upregulation of these genes was not caused by differentiation induction but rather induced by exogenous Foxn1 expression. We also analysed Foxn1 expression by a specific primer pair targeting the 3′-UTR of Foxn124, which enabled us to distinguish exogenous from endogenous Foxn1. Although the influence of exogenous Foxn1 cannot be excluded, significantly upregulated expression of “endogenous Foxn1” indicated that the iPSC-derived cultured cells accurately differentiated into thymic epithelial lineage (Fig. 2c). We also evaluated the expression level of total (endogenous + exogenous) Foxn1 and found that estimated exogenous (total – endogenous) Foxn1 expression level was higher than that of endogenous Foxn1. This might due to sustained strong expression of transduced Foxn1 gene.

iPSC-TECs generate T cells in nude mice recipients

We then examined the functional capacity of iPSC-TECs by transplantation of these cells with immature T-cell progenitors into athymic nude mice (Supplementary Fig. 6a,b). Flow cytometric analysis revealed physiological-like development of CD4 or CD8 single-positive cells derived from co-cultured DN1 cells (Supplementary Fig. 6c). Furthermore, when the differentiated cells were sorted by their expression of EpCAM, we observed a 10-fold increase in the proportion of T cells in peripheral blood from EpCAM+ cell recipient nude mice compared with EpCAM− cell recipients (Fig. 2e). Consistent with this, TCRVβ repertoire expression analysis showed a variety of TCRVβ generation in recipient T cells (Supplementary Fig. 7). These results demonstrated that exogenously expressed Foxn1 exerted a promotive effect on in vitro generation of iPSC-TECs that reconstituted host T cells with a wide TCR repertoire range.

iPSC-TECs contribute to skin graft survival in allogeneic recipients

MHC-homozygous iPSCs are expected to be less immunogenic when transplanted into MHC-homozygous recipients, and the MHC-homozygous iPSCs are banked for the therapeutic use of iPSC-derived grafts25. Thereafter, we evaluated the effect of iPSC-TECs on allograft survival, in which we used skins instead of iPSC-derived therapeutic cells. C3129F1 mice (recipients) received renal subcapsular transplantation of C57BL/6 (B6) iPSC-TEC aggregates following preconditioning of anti-CD4/CD8 antibodies and total body irradiation (Fig. 3a). For control group mice, B6 MEF aggregates were transplanted to assess the possibility of B6 cell influence on recipient “anti-B6” immune responsiveness. Anti-T cell antibody treatment efficiently depleted peripheral CD4+ or CD8+ T cells, and we observed peripheral T cell recovery 5 weeks after the aggregate transplantation (Supplementary Fig. 8). Despite the heterozygous MHC haplotype (H-2b/k) in C3129F1 mice and homozygous haplotype in B6 mice (H-2b), skin grafting from B6 to control C3129F1 caused rapid rejection and resulted in graft loss. In contrast, recipients of iPSC-TEC aggregates showed significantly prolonged survival of B6 skin (Fig. 3b centre panel; median survival time (MST) of 15.5 days). Moreover, skins from third-party BALB/c (H-2d) were rejected independently of pre-transplanted subrenal capsule iPSC-TEC aggregates (Fig. 3b,c; MST = 12 days), and auto skin grafts showed complete engraftment (Fig. 3b; MST > 20 days). The recipients of MEF aggregates rapidly rejected B6 (MST = 12 days) and BALB/c (MST = 12 days) skin graft. These results suggest that iPSC-TEC transplantation reconstitutes the host immune system and specifically extends the survival of allografts from the same donor strain.