Fig 2: Eomes is dispensable for reprogramming of murine fibroblasts. (A) Schematic illustration of the Eomes alleles used in (D–G). MEFs carry one functional null allele with a GFP knock-in at the Eomes locus and a second conditional allele, where exons 2–5 are flanked by loxP sites. The tamoxifen (4-OHT)-inducible CreER-recombinase is expressed from the Rosa26 locus and used to induce the complete genetic deletion of Eomes by 4-OHT administration8. (B,C) 4-OHT treatment regimen used for timed Eomes ablation during reprogramming. Orange lines indicate tamoxifen treatment intervalls: A: d-3 to d-1 (48 h), B: d5–9 (96 h), C: d10-d14 (96 h). (D) Representative images of Alkaline phosphatase staining of iPSC colonies at different timepoints of 4-OHT treatment as indicated. (E,F) FACS-based quantification of (E) Ssea1 and (F) Oct3/4 positive cells at day 20 of reprogramming following with and without 4-OHT administration. Scale bars in all images: 50 µm. Representative experiments from n = 3 in triplicates are shown.

Fig 3: Upregulation of primitive streak and mesendoderm markers during reprogramming of human somatic cells of fibroblast and keratinocyte origin, but absence of EOMES protein. (A) Schematic overview of the reprogramming experiment for the somatic cells of ectodermal –keratinocytes, and mesodermal origin – fibroblasts. (B) Expression patterns of indicated genes during reprogramming of human keratinocytes (upper row) and human fibroblasts (lower row). All mRNA levels are expressed relative to the housekeeping gene HMBS and values have been normalized to iPSCs, which have been set to 1 to illustrate fold induction. (C) Schematic overview of the experimental setup of mesendoderm differentiation. (D) Comparison of marker gene expression for definitive endoderm (CER1) and mesendoderm/primitive streak (LHX1, EOMES) are lower in magnitude for the reprogrammed cells compared to the differentiated cells. (E,F) Protein expression of EOMES and NANOG during the time course of human keratinocytes (E) and human fibroblasts (F) reprogramming. The scale bar represents 100 µm for all images. (G) Western Blot analysis EOMES and NANOG protein expression during reprogramming of fibroblasts. Actin was used as loading control. Lane 1: human foreskin fibroblasts (HFFs) protein lysate, lane 2–6 and 9–15: consecutive days of HFFs reprogramming at indicated time points (d = day), lane 7: fibroblast derived iPSCs, lane 8: mesendodermal differentiation as a positive EOMES control. (H) Mesendodermal differentiation of iPSCs shows a positive signal for EOMES protein.

Fig 4: Confirmation of AT2-like phenotype of hiPSC-derived air–liquid interface (ALI) cultures verified on both protein and mRNA level. (a–l) One-step RT-PCR analysis of different lung cell markers. Statistical comparison between day 27 differentiated alveolar like cell, hiPSC (day 0) and HPAEpiC isolated total RNA. (a) OCT4 pluripotency marker (b–e) expression levels of classical AT2 cell markers. (f) CDH1 as a marker for epithelial cells. (g,h) TM4SF1 and NKX2.1 as alveolar lung progenitor marker. (i,j) Classical markers for alveolar type 1 cells. Median; range [min, max], N = 3; nd = not detectable, ns = not significant, *p < 0.05, **p < 0.01, ****p < 0.0001. (k–m) Immunofluorescence staining of SFTPC at day 27 of differentiation. (k) white: Hoechst33342, green: Phalloidin; (l) red: SFTPC, merged picture; (m) magnification of overlay. (n,o) SFTPB (red) and CDH1 (green) staining in AT2-like cells. Scale bar = 20 µm. (p–r) ABCA3 and (u-w) CDH1 staining in AT2-like cells after 27 days of differentiation. Scale bar = 20 µm. (s,t) Immunofluorescence staining of AT1 marker CAV1 (orange). Scale bar = 20 µm.

Fig 5: Generation of Lung Progenitor Cells (LPCs) following the Definitive Endoderm Formation. (a) Schematic of freezing thawing cycle and subsequent AFE differentiation of previous generated DE cells. (b,c) Relative expression levels during differentiation towards Anterior Foregut Endoderm (AFE) confirmed by the up regulation of the markers SOX2 and FOXA2 within following 4 days. (d–i) Representative immunofluorescence staining for AFE marker expression in hiPSCs following 4 days of directed differentiation (d: bright field, e: SOX2, f: merged bright field and SOX2; g: bright field, h: FOXA2, i: merged bright field and FOXA2). Scale bar = 400 µm. (j–l) Up-regulation of LPC markers NKX2.1, SOX9 and ID2 based on the efficient generation of ventralized AFE (vAFE) cells, within 10 days. (m–o) Representative immunofluorescence staining of the LPC marker NKX2.1 at day 13 (m), day 18 (n) and day 21 (o) of differentiation. Scale bar = 100 µm. (p) Representative FACS plots based on expression of CPM and NKX2.1 (marker for LPCs) in hiPSC-derived LPCs (day 21). Percentages of positive cells are shown in each plot based on isotype control gating strategies. (q) Schematic of differentiation process and essential marker expressions prior to lung progenitor cell banking. Depicted are means ± 95% CI of at least three independent experiments.