Fig. 3. Proximal small intestinal specification following dual-smad inhibition.

a Experimental Schematic of directed differentiation. b Representative micrographs of cells grown in different media conditions at day 34 of differentiation after sorting for NKX2-1^GFP negative cells at day 15 (scale bar = 300 μm, representative of n = 2 independent wells per condition). c Quantification of number of organoids per well at day 50 in different culture conditions. Error bars represent s.e.m. from n = 2 independent wells per condition. d qRT-PCR from day 54 HIOs cultured in either IM + CK, CK + DCI, or IM + CHIR normalized to day 0 hiPSCs and compared with a primary control (human adult colon) (2^−ΔΔCT, technical triplicates normalized to GAPDH or ACTB (β-ACTIN), n = 3 independent differentiations except IM + CHIR (IM + C, n = 2 independent differentiations). Error bars represent the s.d., statistical significance, where indicated, determined by one way-ANOVA followed by Tukey test, *p < 0.05, **p < 0.005, ****p < 0.0001). e Whole mount immunofluorescence of day 60 organoids stained for Villin (VIL), Cdx2 and DNA (blue) (scale bar = 100 μm, representative of n = 3 differentiations).