Supplemental Data Research Design and Methods Differentiation of Human ES Cells Stage 1: Mesoendoderm 60-70% confluent adherent cultures of undifferentiated H1 cells plated on 1:30 Matrigel coated surfaces were exposed to RPMI 1640 medium (Invitrogen) supplemented with 2% fatty acid-free bovine serum albumin (Proliant; Ankeny, IA), 100 ng/ml activin-a (Pepro-tech; Rocky Hill, NJ), and 20 ng/ml of Wnt3A (R&D Systems) for day one only, and 8 ng/ml of FGF2 (Peprotech) for 3-4 days. Stage 2: Endoderm Progenitor Stage 1 cells were exposed to DM-F12 medium (Invitrogen) supplemented with 2% fatty acidfree BSA, 50 ng/ml of FGF7 (Pepro-tech), and 0.25 µm Cyclopamine-KAAD (EMD; Gibbstown, NJ) for two days. Stage 3: Foregut Progenitor Cultures were continued for four days in DM-F12 medium supplemented with 0.25 µm Cyclopamine-KAAD, 2 µm retinoic acid (Sigma-Aldrich; St. Louis, MO), 50 ng/ml of FGF7, 100 ng/ml of Noggin (R&D Systems), and 1% (v/v) B27 (Invitrogen). Stage 4: Endocrine Precursor Stage 3 cells were cultured for three days in DM-F12 medium supplemented with 1 µm ALK5 inhibitor II (Axxora; San Diego, CA), 100 ng/ml of Noggin, 1 µm DAPT (γ-secretase inhibitor; EMD) and 1% B27. Stage 5: Immature endocrine Stage 4 cells were cultured for seven days in DM-F12 medium supplemented with 1% B27 and 1 µm ALK5 inhibitor. Stage 6: Mature endocrine Stage 5 cells were further cultured for an additional seven days in DM-F12 medium supplemented with 1% B27. Clusters were removed from the monolayer by treating with 1X Accutase (Sigma-Aldrich) for 5 min at room temperature. This was followed by aspiration of the enzyme, rinsing with DM-F12 basal media, and gentle scraping of cells using a cell scraper. In some cases, the cell suspension was passed through a 40 µm filter (BD Biosciences; Franklin Lakes, NJ) to retain large clusters. In general, harvested clusters were cultured in DM-F12 medium on non-adherent plates (Corning; Corning, NY) for 1 week in DM-F12, 1% B27 prior to transplantation. In some studies (eg. Supplementary Fig. S3D), harvested clusters were cultured for up to four weeks in DM-F12 + 1% B27. Targeted Screening of Kinase Inhibitor Libraries To identify small molecules that could upregulate endocrine marker expression, we performed a primary screen using EMD kinase inhibitor libraries I and II (Cat# 539744 and 539745; EMD Chemicals; Gibbstown, NJ). The readout for the primary screen was based on qrt-pcr for insulin, glucagon, NeuroD, PDX-1, and NKX2.2 expression. All compounds were administered at a concentration of 1 µmol/l during stage 3 and stage 4 of differentiation. Those compounds that resulted in a 2-fold or greater upregulation of one or more of these genes relative to vehicle control were selected for secondary screening. The six compounds selected for secondary screening were MAPK inhibitor (EMD Cat# 559388), Alk5 inhibitor II (Cat# ALX- 270-445; ENZO Life Sciences; Plymouth Meeting, PA), SMAD3 inhibitor (EMD Cat# 566405),
AKT inhibitor (EMD Cat# 124008), Raf Kinase inhibitor (EMD Cat# 553013), and MEK inhibitor (EMD Cat# 513000). Some experiments were performed using ALK5 inhibitor I (Cat# ALX-270-448; ENZO Life Sciences). Secondary screening data are shown in supplemental figure 2. Flow Cytometry Following release of cells into suspension, cells were washed twice in staining buffer (PBS containing 0.2% BSA). For surface marker staining, 1x10 5-1x10 6 cells were re-suspended in 100 µl blocking buffer (0.5% human gamma-globulin diluted 1:4 in staining buffer). Directly conjugated primary antibodies (CD184 APC (Allophycocyanin)) were added to the cells at a final dilution of 1:20 and incubated for 30 min at 4 o C. For intracellular antibody staining, cells were first incubated with green fluorescent LIVE/DEAD cell dye (Invitrogen) for 30 min at 4 C, followed by a single wash in PBS. Next, cells were fixed in 250 µl of Cytofix/Cytoperm Buffer for 30 min at 4 C followed by two washes in BD Perm/Wash Buffer Solution (both BD Biosciences). Cells were resuspended in 100 µl staining/blocking solution consisting of Perm wash buffer with 2% normal goat serum (or appropriate species of the secondary antibody) and primary antibodies at the appropriate dilutions, incubated for 30 min at 4 C, and washed twice in Perm/Wash buffer before a further 30 min incubation in appropriate secondary antibodies. Antibodies used were as follows: rabbit anti-insulin (1:100; Cell Signaling; Danvers, MA), mouse anti-glucagon (1:1250; Sigma-Aldrich); rabbit anti-synaptophysin (1:100; Dako; Carpinteria, CA); goat anti-rabbit PE (1:200; Invitrogen); goat anti-mouse Alexa 647 (1:500; Invitrogen). Cells were washed twice in Perm/Wash buffer and analyzed using BD FACS Canto. At least 30,000 events were acquired for analysis. Immunocytochemistry hes cells were fixed in 4% PFA followed by two washes in PBS and permeabilized by incubating in 0.5% Triton X-100 for 20 min at room temperature, followed by blocking for 30 min at room temperature in 5% normal serum of the appropriate secondary antibody. Cells were stained overnight at 4 o C with primary antibody diluted in blocking buffer at the appropriate dilution. Primary antibodies used were goat anti-neurod (1:500; R&D Systems), mouse anti- Nkx6.1 (1:500; F55A12; This antibody developed by Ole D. Madsen was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biology, Iowa City, IA 52242), rabbit anti-pdx-1 (1:500; Abcam), and sheep anti-ngn3 (1:500; R&D Systems). Cells were washed 3 times in PBS followed by incubation in the appropriate secondary antibody for 1 h at room temperature. For visualization, cell nuclei were counterstained with Hoeschst or DAPI stain dye in antifade solution (all from Invitrogen). Cells were imaged using a Nikon TE2000 fluorescent microscope. Grafts harvested at d 125 or d 153 were fixed in 4 % paraformaldehyde overnight at 4 C prior to being paraffin embedded and sectioned. All sections were cut at a thickness of 5 µm. Sections were subjected to 10 min heat-induced epitope retrieval (HIER) at 95 C using a 10 mmol/l citrate buffer, ph 6.0. Sections were incubated with mouse anti-glucagon (1:1000; Sigma-Aldrich), goat anti-glucagon (1:50; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), guinea pig anti-insulin (1:1000; Millipore Corp., Billerica, MA), rabbit anti-pc1/3 (1:500; generously provided by Dr. Lakshmi Devi; Mount Sinai School of Medicine, New York, NY), rabbit anti-pc2 (1:500; Affinity BioReagents, Rockford, IL,), mouse anti-somatostatin (1:50; GeneTex, Inc., Irvine, CA), rabbit anti-ghrelin (1:1000; BioVision, Mountain View, CA,), rabbit anti-arx (1:1000; AbCam, Inc., Cambridge, MA), rabbit anti-pdx-1 (1:1000; generously provided by Dr. Joel Habener; Harvard Medical School, Boston, MA), or mouse antiproliferating cell nuclear antigen (PCNA; 1:200; BD Biosciences) as indicated in the text. Visualization of primary antibodies was achieved following incubation with secondary
antibodies conjugated to AlexaFluor 488, 555, 594, or 647 as required (1:1000; Invitrogen) or using the VECTASTAIN ABC kit (Vector Laboratories, Inc., Burlington, ON). Images were captured using a Retiga 2000R LCD camera (QImaging, Surrey, BC) in monochrome and pseudo-coloured (fluorescent images) or in RGB format (DAB images) using OpenLab v5 software (ImproVision, Lexington, MA). In Vivo Analysis of Transplanted Cells Routine blood glucose measurements were taken following a 4 h morning fast. Animals were fasted overnight (16 h) prior to initial blood glucose testing and blood collection for the fast/refeed test. All other metabolic tests were performed following a 4 h morning fast. Intraperitoneal arginine tests were performed via injection of 2 g/kg arginine (Sigma-Aldrich) prepared in saline. For the glucagon tolerance test, 1 µg/kg of glucagon (American Peptide Co.; Sunnyvale, CA) prepared in water was delivered by intraperitoneal injection. For insulin tolerance testing, 0.4 IU/kg of recombinant human insulin (Novolin GE; Novo Nordisk Canada; Mississauga, ON) was delivered by intraperitoneal injection. Where blood glucose levels dropped below 2 mm, a bolus injection of 100 µl 40% glucose was given and mice were subsequently excluded from the analysis. Oral glucose tolerance was tested via gavage of 2 g/kg glucose (40% solution prepared in saline). For all tests, blood glucose was tested via saphenous vein, and saphenous blood samples were collected at the indicated time points using heparinized microhematocrit tubes. Plasma was stored at -20 C and later assayed using a mouse insulin kit (Alpco Diagnostics), a human insulin/glucagon/glp-1 7-36NH2 triplex (Meso Scale Discovery; Gaithersburg, MD), or a glucagon bioassay kit (DiscoveRX; Fremont, CA). To boost the signal of the glucagon bioassay, isobutylmethylxanthine (IBMX) was added to each well at a final concentration of 250 µmol/l. Thirty days (STZ study), 125 or 153 days (studies in normal mice) after transplantation, survival nephrectomy was performed to remove the graft-bearing kidney. Engrafted kidneys and pancreata were fixed in 4% PFA and paraffin-sectioned for subsequent immunocytochemical analysis. qrt-pcr Total RNAs were extracted with the RNeasy Mini Kit (Qiagen; Valencia, CA) and reverse-transcribed using the High Capacity cdna Reverse Transcription Kit (Applied Biosystems, Foster City, CA) according to manufacturer s instructions. cdna (100-120 ng) was amplified by PCR using Taqman Universal Master Mix and Taqman Gene Expression Assays (see table S2 below) which were pre-loaded onto custom Taqman Arrays (all Applied Biosystems). Data was analyzed using Sequence Detection Software (Applied Biosystems) and normalized to undifferentiated human embryonic stem (hes) cells using the Ct method. In some cases part of the graft, harvested at d 125 or d 153 after transplantation, was dissected away from the kidney prior to fixation, and immediately immersed in RNAlater RNA stabilization solution (Qiagen), then incubated at 4 C for 24 h. Total cellular RNA was extracted from the grafts by first further dissecting away kidney tissue, then homogenizing the remaining tissue in QIAzol lysis reagent (Qiagen) using a rotor-type tissue homogenizer. RNA extraction was then performed using the mirneasy kit (Qiagen) with on-column DNase digestion as described in the manufacturer s instructions. cdna synthesis was performed using the RT 2 first strand cdna synthesis kit and mrna levels measured using a custom designed PCR array (SA Biosciences, Frederick, MD) run on a StepOne Plus Real Time PCR machine (Applied Biosystems). Data analysis was performed using the online RT 2 Profiler PCR Array Data Analysis Tool (SA Biosciences). Primers used are listed in Supplementary Table 2. Plasma GLP-1 Measurements
All experiments were approved by the UBC Animal Care Committee. Male Rag1 -/- mice (stock 2216) were obtained from the Jackson Laboratories (Bar Harbor, ME) at 8-10 weeks of age. Mice were maintained on a 12 h light/dark cycle and had ad libitum access to a standard irradiated diet (PicoLab 20; #5058l PMI International; St. Louis, MO). Blood samples were collected from the saphenous vein of conscious restrained mice at the indicated time points using heparinized microhematocrit tubes. Plasma was stored at -20 C and later assayed using a human insulin/glucagon/glp-1 7-36NH2 triplex (Meso Scale Discovery; Gaithersburg, MD). Supplemental Figure 1: Gene Expression Patterns in Differentiating hes Cells (A) Expression of mesoendodermal markers (CD184, GSC, Sox17, Cer, and Foxa2), extraembryonic markers (AFP, Sox7) and mesoderm markers (T) were assessed via qrt-pcr in stage 1 hes cell-derived cells. Data are expressed as mean fold stimulation over undifferentiated H1 cells. n=6. Error bars indicate SE. AFP, α-fetoprotein; CER, cerebus-like; CD184, CXCR4; GSC, goosecoid; T, brachyury. (B) Representative image of hes cell-derived cells expressing SOX17 and HNF3β. Nuclei are labeled with Hoechst 33342. Scale bars, 100 µm. (C) Expression of endodermal and endocrine markers at each differentiation stage (S2 to S6) and in stage 6 clusters (S6C) as measured by qrt-pcr. Data are expressed as fold induction versus adult human islets. n=3 for each stage of differentiation. Error bars indicate SE. (D) Representative images of hes cell-derived cells co-expressing NGN3 and NeuroD at stage 4. Nuclei are labeled with DAPI. Scale bars, 100 µm. (E) Representative images depicting the lack of NKX6.2 expression within PDX-1-expressing hes cell-derived cells at stage 4 of differentiation. Scale bars, 100 µm.
Supplemental Figure 2: Alk5 inhibition improves the efficiency of differentiation to endocrine cells. (A and B) Expression of endocrine markers following treatment with a 1 µmol/l concentration of 6 different kinase inhibitors was assessed via qrt-pcr at stage 3, 4, or 5 as indicated. +/- = compound treatment during stage 3 only; +/+ = compound treatment during stages 3 and 4; -/+ = compound treatment during stage 4 only. Compounds described in supplemental Research Design and Methods. n=2 for each group. (C) Expression of glucagon (green bars) and insulin (red bars) at stage 5 (solid bars) or stage 6 (hatched bars) following treatment with 1 µmol/l Alk5 inhibitor II as indicated. ND, not determined. (D) Endocrine marker expression was assessed via qrt-pcr in stage 4 following treatment with Alk5 inhibitor II at the indicated concentrations. Data are expressed as fold increase versus undifferentiated H1 cells; n=6. (E) Grouped data showing the total number of NGN3-expressing cells per mm 2 assessed at stage 5 following treatment with Alk5 inhibitor II at the indicated concentrations. * P<0.05 vs. 0 µm; P<0.05 vs. 1 µm. n=6.
Supplemental Figure 3: Morphological and subcellular characterization of hes cellderived cells. (A) Representative image showing colocalization of insulin (INS; red) and C-peptide (C-PEP; green) immunofluorescence in stage 6 clusters. Nuclei are labeled with DAPI (blue). Scale bar, 100 µm. (B) Representative image of INS (red), glucagon (GCG; blue), and C-PEP (green) immunofluorescence in stage 6 clusters. Triple-positive cells are visualized as white cells. Scale bar, 100 µm. (C, D) Representative glucagon (GCG; green) and (C) somatostatin (SST; red) or (D) ghrelin (GHR; red) immunofluorescence in adult human islets (left) and stage 6 clusters (right). Nuclei are labeled with DAPI (blue). Scale bar, 100 µm. (E and F) Ultrastructural appearance of Stage 6 clusters (E) and adult human islets (F) was assessed via transmission electron microscopy. Scale bar, 2.5 µm. Blue arrows indicate representative alpha cell secretory granules. Greek lettering indicates individual human islet cell type.
Supplemental Figure 4: hes cell-derived cells produce some GLP-1. (A) Plasma GLP-1 levels during an intraperitoneal arginine challenge (2 g/kg) performed in normal Rag Tm1Mom mice 62 days after transplantation with stage 6 clusters. n=4-7 per group; **p<0.01 versus control. (B) Plasma GLP-1 levels were measured following an overnight fast and after a 45 min refeeding period in normal Rag Tm1Mom mice 99 days after transplantation with stage 6 clusters. n=5-7 per group; **p<0.01 versus respective fasted; ### p<0.001 versus control fasted (Student s t-test).
Supplemental Figure 5: The diabetic milieu does not alter the fate specification of transplanted hes cell-derived cells. (A) Blood glucose in streptozotocin (STZ)-treated diabetic Rag Tm1Mom mice after a 4 h morning fast. Cell Tx mice received STZ (175 mg/kg) at day -8; following diabetes development they were transplanted with stage 6 clusters on day 0 and implanted with slow release insulin pellets (LinBit 1 and 2) as indicated. Linbits were explanted at day 27 and diabetes rapidly returned (n=5-8 animals/group). (B) Blood glucose following intraperitoneal arginine challenge (2 g/kg) performed 28 days posttransplantation and 2 d after Linbit removal (n=8). (C) Plasma glucagon levels in Cell Tx mice during arginine challenge in Panel B. Glucagon levels were below the level of detection (<40 ng/ml) in control mice (n=5-8). Data are expressed as mean ± SE. (D) Grafts were harvested at day 29, paraffin-sectioned and immunostained for glucagon (GCG; green) and insulin (INS; red). A representative image is shown. White arrowhead indicates insulin-positive cell. Scale bar, 100 µm. Merged image includes a DAPI nuclear stain (blue).
Supplemental Figure 6. Alpha cell mass quantification. Pancreata were harvested from control mice and hes cell recipients at d153 post-transplant. (A) Representative image of paraffin-sectioned pancreas immunostained for glucagon (brown). (B) Representative selection masks used to quantify alpha cell mass. Negative pixels appear blue and represent unstained pancreas area. Positive pixels appear red and orange. Scale bars, 50 µm. (C) Quantification of alpha cell mass in control mice and hes cell recipients. Data are plotted as mean ± SE. n=5-7 animals/group; *p<0.05.
Supplemental Table qrt-pcr Analysis of hes Cell Graft Supplemental Table 2. qrt-pcr Primers Applied Gene Biosystems Name Cat. Number AFP Hs00173490_m1 ARX Hs00292465_m1 CER1 Hs00193796_m1 CXCR4 Hs00237052_m1 FOXA2 Hs00232764_m1 GAPDH Hs99999905_m1 GCG Hs00174967_m1 GSC Hs00418279_m1 HNF4A Hs00230853_m1 INS Hs00355773_m1 NEUROD1 Hs00159598_m1 NEUROG3 Hs00360700_g1 NKX2.2 Hs00159616_m1 NKX6.1 Hs00232355_m1 PAX4 Hs00173014_m1 PAX6 Hs00240871_m1 PDX1 Hs00236830_m1 SOX17 Hs00751752_s1 SOX7 Hs00846731_s1 T (Bry) Hs00610080_m1