Supplementary Figure 7: Generation of a human embryonic stem cell based NGN3^GFP reporter line.

(a) The strategy for targeting the NGN3 locus. The NGN3 targeting vector was inserted into the 5’-untranslated region of the NGN3 gene, upstream of the NGN3 start codon (ATG), resulting in an GFP-tagged NGN3 allele. (b) Representative 3D-reconstruction z-stack of hESC-derived EPd3 cells differentiated using protocol A, stained for GFP, EZRIN and DAPI. Right panel represents the culture viewed from the side. (c) Representative image of hESC-derived EPd4 cells differentiated using protocol A, stained for NGN3, GFP and DAPI to show that the reporter is functional. (d) Representative flow cytometry plot of GFP expression of the NGN3^GFP reporter differentiated to EPd2 and EPd9 using protocol A. (e) Percentage (%) of GFP expressing cells in Control (untagged SA121 cells) versus NGN3^GFP cell lines at EPd2-EPd9 differentiated using protocol A. n = 2 except d9 n = 1. (f) qPCR analysis of GFP from PEd11-EPd10 cultures of human embryonic stem cells differentiated to endocrine cells using protocol A, or S3d7-S5d13 cultures differentiated using protocol B. n = 1. (g) qPCR analysis of NGN3 from PEd11-EPd10 cultures of human embryonic stem cells differentiated to endocrine cells using protocol A, or S3d7-S5d13 cultures differentiated using protocol B. n = 1. (h) qPCR analysis of INS from PEd11-EPd10 cultures of human embryonic stem cells differentiated to endocrine cells using protocol A, or S3d7-S5d13 cultures differentiated using protocol B. n = 1. (i) Ngn3^High/NGN3^High and Ngn3^Low/NGN3^Low cells were isolated using flow cytometry from E15.5 Ngn3^{Y FP+} pancreata or hESC-derived NGN3+ cells (EPd3—Protocol A). Expression of Ngn3, Muc1, Sox9, Hes1, Pdx1, Isl-1 and NeuroD was determined by qPCR and normalized to β-actin. Mouse: n = 5 extracts of RNA (from 1 pancreata each), from three independent experiments, Ngn3 p_TT = 0.0414, Muc1 p_TT = 0.0066, Sox9 p_TT = 0.0289, Hes1 p_TT = 0.0286, Pdx1 p_TT = 0.7503, Isl-1 p_TT = 0.0106, NeuroD p_TT = 0.0411; Human: n = 3 extracts of RNA (from 1 differentiated plate each), from three independent differentiations, NGN3 p_TT = 0.0127, MUC1 p_TT = 0.0461, SOX9 p_TT = 0.0102, HES1 p_TT = 0.0117, PDX1 p_TT = 0.2470, ISL1 p_TT = 0.0184, NEUROD p_TT = 0.0301. Error bars represent ± SEM. Scale bar, 100 μm (b), 20 μm (c). Statistic source data can be found in Supplementary Table 3. Statistical analysis: two-tailed student’s t-test (p_TT).