Supplementary Figure 1.

Transcription

1 Supplementary Figure 1. (A) UCSC Genome Browser view of region immediately downstream of the NEUROG3 start codon. All candidate grna target sites which meet the G(N 19 )NGG constraint are aligned to illustrate the flexibility of CRISPR/Cas9 targeting. The sequences chosen for targeted mutagenesis are highlighted in red. (B) A list of all genotypes and sequences from expanded subclones illustrating all identified insertions and deletions (INDELs). The clone is indicated by the first letter/number (C1-C8 for grna#1) (A5, B2, C2, C5 for grna#2). The wild type (WT) allele and alleles with INDELs are indicated, with the insertions (red) or deletions (-) in NEUROG3 indicated on the right. Some genotypes are only predicted using the Mixed Sequence Reader (indicated by asterisk, see supplemental methods). Genotypes for all cell lines used for differentiation experiments were confirmed by subcloning and Sanger sequencing and are indicated as wild type (WT), heterozygous (+/-) or homozygous (-/-) loss-of-function for NEUROG3. (C-E) Analysis of expanded clones. All cell lines exhibited normal morphology (C), expressed markers of pluripotency such as NANOG and OCT4 (D), and had normal karyotypes (E) (only NEUROG3 -/- clone C3 is shown as an example). (F) Predicting low off target guide RNAs using the CRISPR Design Tool ( The guide RNA sequences used for NEUROG3 have no predicted matches to any other sites in the genome. The most similar sites, which had 3 predicted mismatches with both grna-1 and grna-2 are highlighted in red. Each off-target site is classified as nongenic or intronic, no off-target sites were located in exons. Scale bars = 50 μm.

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3 Supplementary Figure 2. (A) Analysis of efficiency of directed differentiation of hescs into pancreatic progenitors at different stages of differentiation. The efficiency of definitive endoderm differentiation was assessed by co-expression of SOX17 and FOXA2, posterior foregut by PDX1 expression, and pancreatic precursors by NKX6.1 expression. Endocrine differentiation was indicated by expression of Chromogranin A (CHGA). Images are representative of >10 separate experiments. (B) Differentiation into pancreatic precursors is very efficient with >95% of monolayer cultures expressing PDX1, and 58% of cells co-expressing NKX6.1. 4% of cells expressed CHGA as assessed by high content tile-scan imaging. (C) Nuclear NKX6.1 expression levels for NEUROG3 +/+, NEUROG3 +/-, and NEUROG3 -/- day 12 cultures were compared by immunofluorescence. (D) Example of a hi-res confocal tile-scan of day 12 pancreatic precursors illustrating how data were collected for high-content imaging and quantitative analysis. PDX1 staining was uniform across the entire monolayer. Analysis of CHGA expression showed reduction of endocrine cells in NEUROG3 +/- lines and complete absence in NEUROG3 -/- lines. To generate a NEUROG3 reporting hesc line, the 5.5kb promoter region of NEUROG3 was cloned upstream of a fluorescent protein mcherry. The NEUROG3 reporter construct was stably transduced into H1 stem cells using a lentiviral vector. (E) Colocalization and quantitation (F) of mcherry fluorescence and immunostaining for NEUROG3 in day 12 pancreatic precursors. Approximately 90% of cells expressing mcherry also express NEUROG3 protein, indicating faithful expression. Just over 50% of NEUROG3+ cells express the reporter mcherry indicating that the reporter is restricted to NEUROG3-expressing cells, but is not at high levels in all NEUROG3 cells. (G) NEUROG3-mCherry expressing cells were collected from day 12 pancreatic precursors by FACS and compared to NEUROG3-negative cells. mrna levels of transcription factors and endocrine markers in NEUROG3- mcherry positive cells were measured by qpcr and were normalized to expression levels in NEUROG3-negative cells. (H) Day 12 pancreatic precursors were stained for INS, GCG, and SST (see Fig. 2B,B ) and quantified by high content image analysis (5x5 tile scan). The number of cells expressing each hormone in both NEUROG3 +/+ and NEUROG3 -/- cultures is shown (NEUROG3 -/- did not have any detectable hormone expression). The area of the circles represent the relative ratios of hormone expression. Overlapping areas of the circles represent polyhormonal cells (i.e. the yellow area illustrates the fraction of cells expressing both INS and GCG). Scale bars = 100 μm.

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5 Supplementary Figure 3. Analysis of pancreatic and endocrine cell transcription factors by qpcr in NEUROG3 +/+, NEUROG3 +/-, and NEUROG3 -/- lines generated from grna-1 (A) and grna-2 (B). All lines were analyzed at the pancreatic progenitor stage (day 12). Data are represented as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001.

6 Supplementary Figure 4. (A) Engraftment of hesc-derived pancreatic progenitors into the splenic lobe of the pancreas of immune-deficient NOD-Scid IL-2R null (NSG) mice. (B) A hi-resolution confocal tile-scan of engrafted progenitors from NEUROG3 +/+, NEUROG3 +/-, NEUROG3 -/- lines 6 weeks after transplantation. Staining for insulin (INS), glucagon (GCG) and somatostatin (SST) detects beta, alpha, and delta cells in the pancreas. Human cells were distinguished from mouse islets with an antibody that recognizes a human nuclear antigen (HNUC) and is outlined with yellow dots. (C) The number of cells expressing INS, GCG, and/or SST was assessed by high content image analysis of NEUROG3 +/+ and NEUROG3 +/- transplants. The area of each circle represents the total cell counts from representative sections (n 3) of 6-week old transplants (n=3). Overlapping areas of the circles represent polyhormonal cells (i.e. the yellow area illustrates the fraction of cells expressing both INS and GCG). (D) Nuclear PDX1 and (E) NKX6.1 fluorescence were quantified for representative NEUROG3 +/+ and NEUROG3 +/- 6-week transplants. Scale bars = 100 μm.

7 Supplementary Table 1. List of primary antibodies used in this study. Antigen Animal Company, Cat # Concentration CHGA rabbit Immunostar # :500 FoxA2 goat Santa Cruz sc6554 (M-20) 1:500 Glucagon rabbit Zymed 1:500 HNUC Mouse Chemicon MAB :1000 INS guinea pig DAKO AO564 1:1000 NANOG rabbit Cell Signaling :1000 NEUROG3 mouse DSHB 1:100 Nkx6.1 mouse DSHB F55A10 1:100 Oct4 mouse Santa Cruz sc :500 Pdx1 goat abcam ab :5000 Somatostatin goat sc :1000 Sox17 goat R&D 1:500 Supplementary Figure 2. List of primers used in this study. qpcr Primers Gene Forward Reverse ARX CTGCCTTCTCCCGCTTG CACTACCCGGACGTCTTCAC CHGA TGTGTCGGAGATGACCTCAA GTCCTGGCTCTTCTGCTCTG GCG CAGCAAGTATCTGGACTCCAGG CCAGTTTATAAAGTCCCTGGC IA1 ACACAACGTAAAAGTGGGGG GAAAGTGTCGTCTCCGCTTC INS GAACCAACACCTGTGCGGCTCA TGCCTGCGGGCTGCGTCTAGT MYT1 CCGTGTGTCCACCTCTGATT TTCATGATTGCTTTCCGTGA NEUROD ATCAGCCCACTCTCGCTGTA GCCCCAGGGTTATGAGACTAT NEUROG3 CGCCGGTAGAAAGGATGAC GACGTGGGGCAGGTCACTT NKX2.2 GGAGCTTGAGTCCTGAGGG TCTACGACAGCAGCGACAAC NKX6.1 CGAGTCCTGCTTCTTCTTGG GGGGATGACAGAGAGTCAGG OCT4 GTGGAGGAAGCTGACAACAA CTCCAGGTTGCCTCTCACTC PAX4 TGTGCAGAGATGATTCCTGG GAGGGTCTGGTTTTCCAACA PDX1 CGTCCGCTTGTTCTCCTC CCTTTCCCATGGATGAAGTC PTF1A AGAGAGTGTCCTGCTAGGGG CCAGAAGGTCATCATCTGCC RFX6 CCAGTTTTTGAGCTAAGCGAA TGGCATCAAAGAGAGCAGTG SOX17 GGCGCAGCAGAATCCAGA CCACGACTTGCCCAGCAT Genotyping NEUROG3 Genotyping CGGTCGTTGGCCTTCTTTCG CCACCTAGCCTCGGAATCG