Fig 2: Characterization of ventral midbrain dopaminergic neurons differentiated from the TH-mOrange reporter iPSC line. (A) Scheme depicting iPSC differentiation towards floor plate specific ventral midbrain dopaminergic neurons (vmDAn). (B) Immunofluorescence analysis of representative floor plate progenitors from SP_11 TH reporter iPSCs after 13 days of ventral midbrain patterning staining positive for the floor plate markers FOXA2 and EN1 (red), LMX1A and NESTIN (green). Scale bar, 50 µm. (C) Live imaging of differentiated TH-mOrange iPSC cell line towards floor plate vmDAn, showing fluorescent neuronal cells after 50 days of differentiation. (D) Immunofluorescence analysis of DAn differentiated for 50 days and stained positive for tyrosine hydroxylase (TH; green), mOrange (red) and MAP2 (grey). Inset show in detail double-labeled mOrange+/TH+ DAn as well as double negative mOrange-/TH- MAP2+ non-DAn (empty arrowhead) confirming faithful reporter activity. Nuclei were counterstained with DAPI. Scale bars 50 µm and 10 µm for the large images and the insets respectively. (E) Immunofluorescence analysis of DAn differentiated for 50 days and stained for TH and FOXA2 (red and green respectively; left) or GIRK2 and TH (red and green respectively; right). Nuclei were counterstained with DAPI. Scale bar, 10 µm both main image and insets. (F) Quantitative analysis of the percentage of cells differentiated from either original SP_11 or TH-mOrange iPSC lines stained positive for TH. (G) Percentage of TH+ cells stained positive for GIRK2 or FOXA2.

Fig 3: Class I and class II enhancers can be distinguished in liver and lung development.a and b Schematic of stepwise differentiation of hESCs to hepatic progenitors (HP) (a) and induced human pluripotent stem cells (iPSC) into alveolosphere organoids (ALV) (b). AFG, anteriorized foregut. Select growth factors for hepatic (a) and alveolar (b) lineage induction are indicated. FGF2, fibroblast growth factor 2; BMP4, bone morphogenic protein 4; CHIR, CHIR99021; RA, retinoic acid. c Heatmap showing density of FOXA1 and FOXA2 ChIP-seq reads at hepatic enhancers in GT and HP. Heatmap is centered on FOXA1 and FOXA2 peaks, respectively, and spans 5 kb. Hepatic enhancers are classified based on temporal pattern of FOXA1 and FOXA2 occupancy. d Heatmap showing density of FOXA1 ChIP-seq reads at alveolar enhancers in AFG and ALV. Heatmap is centered on FOXA1 peaks and spans 5 kb. Alveolar enhancers are classified based on temporal pattern of FOXA1 occupancy. e Genome browser snapshots showing FOXA1 and FOXA2 ChIP-seq signal at a class I hepatic enhancer near AAT and a class II hepatic enhancer near CEBPA in GT and HP. f Genome browser snapshots showing FOXA1 ChIP-seq signal at a class I alveolar enhancer near SOX2 and a class II alveolar enhancer near NKX2.1 in AFG and ALV. g and h Enriched de novo transcription factor (TF)-binding motifs at class I against a background of class II enhancers and vice versa for hepatic (g) and alveolar enhancers (h). Fisher’s exact test, 1-sided, corrected for multiple comparisons. All ChIP-seq experiments, n = 2 replicates from independent differentiations.

Fig 4: Differentiation of Capacitated Naive iPSCs(A) Flow cytometry analysis of SOX17 and CXCR4 expression after 3 days definitive endoderm induction of primed S6EOS and capacitated niPSC4 cells.(B) Immunostaining for FOXA2 and SOX17 after 3 days definitive endoderm induction of niPSC2.(C) qRT-PCR analysis of definitive endoderm markers after 3 days induction of niPSC2.(D) Immunostaining for SOX1 and PAX6 after 10 days neuroectoderm induction of niPSC2.(E) qRT-PCR analysis of neuroectoderm marker expression.(F) Immunostaining for TBX6 after 6 days of paraxial mesoderm induction of niPSC2.(G) qRT-PCR analysis of paraxial mesoderm markers.Scale bars, 100 µm. Error bars indicate SD of three technical replicates.See also Figure S4.

Fig 5: hES-derived VM progenitors grown on bacterial cellulose express markers of dopaminergic lineage. VM progenitors grown on laminin coated bacterial cellulose (Lam-coat) express FOXA2, LMX1, and FOXA2 and are colocalised with LMX1. The expression of FOXA2 is comparable to the plastic substrate controls (Lam-wells). The expression of LMX1 and LMX1 with FOXA2 is decreased compared with cells cultured on plastic. The proportion of cells showing colocalisation of a nuclear marker, Hoechst 33,342 (blue) with the transcription factor A FOXA2 (magenta); B LMX1 (yellow); C FOXA2 (magenta) and LMX1 (yellow). Two-tailed unpaired Student’s t test were used for statistical analysis. ***P < 0.001. Experiments were performed from N = 3 experimental repeats with n = 3 technical repeats. All data are plotted with mean values ± standard deviation normalised to Laminin coated plastic (Lam-wells). Scale bar: 30 µm