An organized and functional thymus generated from FOXN1-reprogrammed fibroblasts

Abstract

A central goal of regenerative medicine is to generate transplantable organs from cells derived or expanded in vitro. Although numerous studies have demonstrated the production of defined cell types in vitro, the creation of a fully intact organ has not been reported. The transcription factor forkhead box N1 (FOXN1) is critically required for development of thymic epithelial cells (TECs), a key cell type of the thymic stroma. Here, we show that enforced Foxn1 expression is sufficient to reprogramme fibroblasts into functional TECs, an unrelated cell type across a germ-layer boundary. These FOXN1-induced TECs (iTECs) supported efficient development of both CD4(+) and CD8(+) T cells in vitro. On transplantation, iTECs established a complete, fully organized and functional thymus, that contained all of the TEC subtypes required to support T-cell differentiation and populated the recipient immune system with T cells. iTECs thus demonstrate that cellular reprogramming approaches can be used to generate an entire organ, and open the possibility of widespread use of thymus transplantation to boost immune function in patients.

Figures

Figure 1. Enforced Foxn1 expression induces epithelial identity in fibroblasts

a, iFoxn1 transgene. b, Schematic showing procedure for generating iFoxn1 MEFs. c, GFP expression in iFoxn1 and Rosa26CreERt2/+ (control) MEFs 10 days after 4OHT treatment. d,Foxn1 mRNA expression in iFoxn1 and Rosa26CreERt2/+control MEFs 10 days after 4OHT treatment, and E15.5 wild-type EpCAM+ fetal TEC, normalized to the geometric mean of two housekeeping (2HK) genes. Data shown are mean ± s.d. from n=3 independent experiments. e, Bright field and immunofluorescence images showing morphology and Keratin 8 staining, 10 days after 4OHT treatment. Scale bar, 100μm. f, EpCAM and GFP expression 10 days after 4OHT treatment, after gating on live cells. Values shown are mean ± s.d. from n=3 independent experiments. g-i, mRNA expression levels of the genes shown in purified EpCAM+ iFoxn1 and control MEFs normalized to the geometric mean of 2HK. Data shown are mean ± s.d. from n=3 independent experiments (ns, not significant; *p<0.05). g, Expression level is shown relative to E15.5 EpCAM+ fetal TEC or skin. Blue bars, Rosa26CreERt2/CAG-Foxn1-IRES-GFP (iFoxn1); grey bars, Rosa26CreERt2/+ (control); black bars, fetal TEC, throughout. n≥3 independent experiments for panels (c-i). (d,g-i) 3 technical replicates were run for each n. cDNA was generated from 200 cells using the cells direct (amplification) method (d,g,h) or from 50,000 cells without amplification (i). Error bars or values show mean ± s.d. See also Supplementary Fig. 1 and Supplementary Fig. 2.

Figure 2. iTEC can support T cell development in vitro.

a, Strategy for in vitro T cell differentiation assay. Monolayers of iTEC or control MEFs, 7 days following 4OHT treatment, were seeded with ETP (Lin-CD25−C-Kit+) isolated from E14.5 wild-type thymus lobes and were analyzed 12 days later (Lin: CD3ε, CD4, CD8, CD11b, CD11c, B220, Gr-1, NK1.1, Ter119). Right panel, bright field image of iTEC/thymocyte co-culture. Scale bar, 50μm. b-d, Plots show staining after gating on CD45+ cells following 12 days co-culture with iTEC or control MEFs. Values shown are mean ± s.d. from n=4 independent experiments. d, TCRβ expression on CD4+ and CD8+ cells from iTEC co-cultures or wild-type (WT) thymus. e, Control MEFs or iTEC after gating on CD45- cells, and EpCAM+ cells for iTEC, following 12 days co-culture with thymocytes. Values shown are mean ± s.d. from n=3 independent experiments. See also Supplementary Fig. 3 and Supplementary Fig. 4.

Figure 3. iTEC form a functional thymus in vivo.

a, Schematic of grafting assay. iTEC (5 days following Foxn1induction) or control MEFs were aggregated with wild-type CD45+Lin− thymocytes and E12.5-13.5 wild-type CD45−PDGFRαβ+ thymic mesenchymal cells, and grafted under the kidney capsule of adult mice. Lin: CD3ε, CD4, CD8, CD11b, CD11c, B220, Gr-1, NK1.1, Ter119. b, Summary of recovered grafts. c, Haematoxylin and eosin staining, and pan-cytokeratin (panK) staining of iTEC-derived kidney graft. m, medulla; c, cortex; scale bar, 500μm. d–e, Analysis of iTEC grafts, and wildtype (WT) thymi from 8 week old mice. e, shows CD45−EpCAM+ cells from gate defined in (d); GFP expression reports CAG-iFoxn1. Data shown are representative and are from one of at least 3 independent experiments.

Figure 4. Intrathymic T cell development in iTEC-derived grafts

a,b, Immunohistochemical analyses of iTEC grafts and wild-type (WT) thymi using cTEC (CD205 and β5t) and mTEC (Keratin 14 (K14) and UEA-1) specific markers, and α-AIRE. Scale bars (a), 150μm; scale bar (b), 25μm. c, Flow cytometry of MHC Class II expression for CD45-EpCAM+ cells defined in (Fig. 3d). Gates show MHC Class IIlo and MHC Class IIhi populations defined using WT TEC. (a–c) Data shown are representative and are from one of at least 4 independent experiments. (d) RT-qPCR analysis of 50 CD45-EpCam+MHC Class II+ iTEC, recovered from a single graft 7 weeks post-transplantation, for the markers shown. Data are shown relative to expression in E15.5 WT total EpCam+ TEC after normalization to 2HK (HMBS and TBP); expression level in E15.5 WT is 1 for all samples. Values shown are from 1 experiment. (e–h) Analysis of 8 week-old WT mouse thymus, iTEC grafts or cells collected from control MEFs graft site at 4 weeks post-transplantation. (f,g,h) show thymocyte populations defined in (e). e, CD4 and CD8 expression after gating on CD45+ cells. f, Percentage of CD4+CD8+ cells. g, TCRβ expression. h, TCRγδ expression on CD4-CD8- thymocytes. (e,g) Data shown are representative and are from one of at least 4 independent experiments, (f) each data point represents a separate graft from n=4 independent experiments, (h) data shown are from 1 experiment.

Figure 5. A diverse and functional peripheral T cell repertoire is generated by iTEC-derived grafts

a, Schematic showing experimental design for (b-i). (b-f) Representative analysis of spleen and lymph node cells or peripheral blood (PB) from nude mice, using the T cell markers indicated. T cells were detected in the PB of two of three iTEC recipients. Splenocytes (b,d) or peripheral blood (c,e,f) from 6 weeks (f), 8 weeks (e), 14 weeks (b,d) or 2-weekly intervals, as shown (c) post-transplantation. (b,d,e) Data shown are representative and are from one of at least 3 independent experiments, (c) values shown are mean from n=2 independent experiments. Values used to derive mean in (c): iTEC graft recipient mice at 0 weeks, 0; 4 weeks, 1.03 and 0.61; 6 weeks, 1.40 and 1.10; 8 weeks, 2.02 and 1.46, and control graft recipient mice at 0 weeks, 0; 4 weeks, 0.02 and 0.09; 6 weeks, 0.07 and 0.19; 8 weeks, 0.22 and 0.15. (f) data shown are from 1 experiment. (g) Analysis of CD3+ cells from pooled spleen and lymph nodes of recipient nude mice 20 weeks post-transplantation. Data shown are representative and are from one of 2 independent experiments,. (h,i) Analysis of IL2 expression in CD4+ cells from lymph nodes of recipient nude mice 20 weeks post-transplantation. Values represent mean ± s.d. for cells from n=3 separate culture dish wells from 1 experiment. Values used to derive mean and s.d. in (i): wildtype, 55, 33, 75 (unstimulated) and 700, 931, 898 (stimulated); iTEC, 61, 22, 42 (unstimulated) and 572, 403, 780 (stimulated); control, 11, 20, 8 (unstimulated) and 50, 27, 65 (stimulated). See also Supplementary Fig. 5.