Cell Culture 2 , and 100% humidity. We have not performed these cultures in alternative suspension culture systems or in planar tissue culture; such a modification would likely require significant modification to the cell culture protocol described here. The human embryonic stem cell line HUES8 and two hiPSC cell lines (hiPSC-1 and hiPSC-2) were utilized for all experiments shown. HUES8 cells are part of the NIH human embryonic stem cell registry eligible for NIH funding and differentiate well into multiple lineages ( Chetty et al., 2013 Chetty S.

Pagliuca F.W.

Honore C.

Kweudjeu A.

Rezania A.

Melton D.A. A simple tool to improve pluripotent stem cell differentiation. Undifferentiated hPSC lines were maintained in mTeSR1 (StemCell Technologies; 05850) in 500ml spinner flasks (Corning, VWR; 89089-814) placed on a 9-Position stir plate (Chemglass) set at rotation rate of 70rpm in a 37°C incubator, 5% CO, and 100% humidity. We have not performed these cultures in alternative suspension culture systems or in planar tissue culture; such a modification would likely require significant modification to the cell culture protocol described here. The human embryonic stem cell line HUES8 and two hiPSC cell lines (hiPSC-1 and hiPSC-2) were utilized for all experiments shown. HUES8 cells are part of the NIH human embryonic stem cell registry eligible for NIH funding and differentiate well into multiple lineages (). hiPSC-1 was derived using retroviral reprogramming with three factors (OCT4, SOX2, KLF4), and hiPSC-2 was derived using Sendai viral reprogramming with four factors (OCT4, SOX2, KLF4, c-MYC). Cells were adapted to grow in suspension by seeding 1-2 million cells per ml in mTESR1 in a spinner flask and split for at least five passages before testing differentiation capacity. Cells were dispersed with Accutase (StemCell Technologies) and seeded as single cells at 0.5 million cells per ml in mTESR1 with 10μM Y27632 (Abcam; ab120129). mTESR1 media was changed (without Y27632) 48 hr later. Cultures were split 24 hr after that media change to keep cluster diameter size < 300μm and prevent necrosis or spontaneous differentiation. Cell lines with different doubling times may require different seeding concentrations or time in pluripotency media to expand. Cultures were regularly tested for pathogens, karyotype and for maintenance of pluripotency markers. Media used for directed differentiation were as follows: S1 media: MCDB131 (Cellgro; 15-100-CV) + 8mM D-(+)-Glucose (Sigma; G7528) + 2.46 g/L NaHCO 3 (Sigma; S3817) + 2% FAF-BSA (Proliant; 68700) + ITS-X (Invitrogen; 51500056) 1:50.000 + 2mM Glutamax (Invitrogen; 35050079) + 0.25mM Vitamin C (Sigma Aldrich; A4544) + 1% Pen/Strep (Cellgro; 30-002-CI). S2 media: MCDB131 + 8mM D-Glucose + 1.23 g/L NaHCO 3 + 2% FAF-BSA + ITS-X 1:50.000 + 2mMl Glutamax + 0.25mM Vitamin C + 1% Pen/Strep. S3 media: MCDB131 + 8mM D-Glucose + 1.23 g/L NaHCO 3 + 2% FAF-BSA + ITS-X 1:200 + 2mMl Glutamax + 0.25mM Vitamin C + 1% Pen/Strep. S5 media: MCDB131 + 20mM D-Glucose + 1.754 g/L NaHCO 3 + 2% FAF-BSA + ITS-X 1:200 + 2mM Glutamax + 0.25mM Vitamin C + 1% Pen/Strep + Heparin 10μg/ml (Sigma; H3149). S6 media: CMRL 1066 Supplemented (Mediatech; 99-603-CV) + 10% FBS (HyClone, VWR; 16777) + 1% Pen/Strep. All media were filter sterilized through a 0.22μm bottle top filter Filter (Corning). For all following media changes, small molecules and growth factors were added to the base media immediately before media change in a low-light hood. Schulz et al., 2012 Schulz T.C.

Young H.Y.

Agulnick A.D.

Babin M.J.

Baetge E.E.

Bang A.G.

Bhoumik A.

Cepa I.

Cesario R.M.

Haakmeester C.

et al. A scalable system for production of functional pancreatic progenitors from human embryonic stem cells. Rezania et al., 2012 Rezania A.

Bruin J.E.

Riedel M.J.

Mojibian M.

Asadi A.

Xu J.

Gauvin R.

Narayan K.

Karanu F.

O’Neil J.J.

et al. Maturation of human embryonic stem cell-derived pancreatic progenitors into functional islets capable of treating pre-existing diabetes in mice. Hrvatin et al., 2014 Hrvatin S.

O’Donnell C.W.

Deng F.

Millman J.R.

Pagliuca F.W.

DiIorio P.

Rezania A.

Gifford D.K.

Melton D.A. Differentiated human stem cells resemble fetal, not adult, β cells. Using significant modifications of previously published protocols (), populations of DE, PGT and PP1 cells were generated as described below. For initiation of SC-β cell differentiation, HUES8, hiPSC-1, or hiPSC-2 cells were seeded at 0.6 million cells/ml (for example, 150 million cells in 250 ml mTeSR1 media + 10μM Y27632). The differentiation was started 48 hr later for HUES8 and 72 hr later for hiPSC-1 and hiPSC-2 by changing media to Day 1 media. Media changes were as follows – Day 1: S1 + 100ng/ml ActivinA (R&D Systems; 338-AC) + 3μM Chir99021 (Stemgent; 04-0004-10). Day 2: S1 + 100ng/ml ActivinA. Days 4, 6: S2 + 50ng/ml KGF (Peprotech; AF-100-19). Days 7, 8: S3 + 50ng/ml KGF + 0.25μM Sant1 (Sigma; S4572) + 2μM Retinoic acid (RA) (Sigma; R2625) + 200nM LDN193189 (only Day 7) (Sigma; SML0559) + 500nM PdBU (EMD Millipore; 524390). Days 9, 11, 13: S3 + 50 ng/ml KGF + 0.25μM Sant1 + 100nM RA. Days 14, 16: S5 + 0.25μM Sant1 + 100nM RA + 1μM XXI (EMD Millipore; 565790) + 10μM Alk5i II (Axxora; ALX-270-445) + 1μM L-3,3′,5-Triiodothyronine (T3) (EMD Millipore; 64245) + 20ng/ml Betacellulin (Thermo Fisher Scientific; 50932345). Days 18, 20: S5 + 25nM RA + 1μM XXI + 10μM Alk5i II + 1μM T3 + 20ng/ml Betacellulin. Days 21-35 (media change every second day): S6 + 10μM Alk5i II + 1μM T3. In the final stage, cells were analyzed between days 28 and 33 of the protocol. Only cell preparations that had the following fraction of cells with the indicated marker at the indicated time in the protocol were considered: > 95% OCT4+ on day 1, > 90% SOX17+ on day 4, > 85% PDX1+ on day 9, > 40% PDX1+/NKX6-1+ on day 14, and > 20% C-peptide+/NKX6-1+ on day 28. Furthermore, cultures were discarded if loss of cell cluster integrity was observed, as indicated by shrinking cluster diameter or high levels of single cells in the supernatant. Rezania et al., 2012 Rezania A.

Bruin J.E.

Riedel M.J.

Mojibian M.

Asadi A.

Xu J.

Gauvin R.

Narayan K.

Karanu F.

O’Neil J.J.

et al. Maturation of human embryonic stem cell-derived pancreatic progenitors into functional islets capable of treating pre-existing diabetes in mice. For generation of PH protocol cells, HUES8 was used, and the same differentiation protocol was followed until day 14. On days 14 and 16, cells were fed with S3 + 1μM Alk5i II, 200nM LDN193189, and 100nM RA (). On days 18 and onward, cells were fed every other day with S3 + 100nM RA. Cells were maintained in this media until experimental analysis. Cells were maintained in culture and tested after the same number of days in differentiation media as the SC-β cells to control for the impact of time in culture.

Glucose-Stimulated Insulin Secretion Krebs buffer (Krb) was prepared as follows: 128 mM NaCl, 5 mM KCl, 2.7 mM CaCl 2 , 1.2 mM MgCl 2 , 1 mM Na 2 HPO 4 , 1.2 mM KH 2 PO 4 , 5 mM NaHCO 3 , 10 mM HEPES (Life Technologies; 15630080), 0.1% BSA (Proliant; 68700) in deionized water. Krb solutions containing 2mM glucose (low glucose), 20mM glucose (high glucose), or 2mM glucose and 30mM KCl (KCl polarization challenge) were prepared and equilibrated to 37°C. Human islets (approximately 500 IEQ, Prodo Laboratories) or differentiated PH or SC-β cells (approximately 500,000 cells sampled from cultures between days 28-35 of differentiation) were sampled. These clusters (islets or stem cell-derived cells) were washed twice with 1 ml Krb buffer and then preincubated in 3 ml low glucose Krb for two hours to remove residual insulin. Note: for all incubations tube lids were left open and covered by a lid that allowed for air exchange. Clusters were washed 2 times in Krb and then incubated in 1 ml low glucose Krb for 30 min. A sample of 200ul of the supernatant was collected after incubation for ELISA analysis (low glucose sample). Clusters were washed 2 times in Krb and then incubated in high glucose Krb for 30 min and 200ul of supernatant was collected after incubation (high glucose sample). Challenging with low and high glucose was repeated two additional times (3 paired challenges in total for each differentiation batch or islet donor). Finally, clusters were washed twice in Krb and then incubated in Krb containing 2mM glucose and 30mM KCl (polarization challenge) for 30 min. A sample of 200ul of the supernatant was collected after incubation for ELISA analysis (KCl polarization challenge sample). After the KCl challenge, clusters were dispersed into single cells using TrypLE Express (Life Technologies; 12604-013) and cell number was counted automatically by a Vi-Cell (Beckman Coulter). Supernatant samples containing secreted insulin were processed using the Human Ultrasensitive Insulin ELISA (ALPCO Diagnostics; 80-INSHUU-E01.1) and samples were measured by a FLUOstar optima spectrophotometer (BMG lantech) at 450 nm. If the ELISA was not preformed on the same day, samples were stored at −80°C. Insulin concentrations in samples collected for each challenge were normalized by the cell number in each sample, as measured by the Vi-Cell automated counting.

Proinsulin and Insulin Content Measurements Collected cells were settled to the bottom of a tube and the supernatent removed. The cell pellets were placed in 1 ml of 1.5% HCl in 70% ethanol and stored at −20°C for 24 hr. Cell pellets were then vortexed briefly and stored at −20°C for an additional 24 hr. Tubes were then centrifuged at 2100 RCF for 15 min. The 1 ml supernatant was then transferred to a new tube and neutralized by addition of 1 ml of 1M TRIS (pH 7.5). The neutralized supernatant containing the extracted proinsulin and insulin was then measured using intact human proinsulin (BioVendor; RZ193094100) and human Ultrasensitive Insulin ELISA kits.

Calcium Imaging Each well of a 96-well plate was treated with a 50ul of hESC-qualified Matrigel (BD Biosciences) that had been diluted 1:75 in DMEM:F12 (Invitrogen). The plate was incubated for 60 min in the incubator before the excess matrigel solution was aspirated off to leave a thin layer of extracellular matrix to promote cell attachment. Human islet clusters or hPSC-derived differentiated clusters (approximately 20 clusters or islets per well) were resuspended in 100 ul of CMRL supplemented media and added to these coated wells to incubate for 24 hr. After incubation and attachment, the wells were washed with prewarmed (37°C) Krb buffer containing 2.5mM glucose. The clusters were then incubated with 50μM Ca2+-sensitive fluorescent probe Fluo4-AM (Life Technologies; F14217) in 2.5mM glucose Krb buffer for 45 min in a 37°C incubator. Clusters were washed with 2.5mM glucose Krb buffer then incubated further in 37°C incubator for additional 15 min. Clusters were then immediately staged on an AxioZoom V16 microscope (Carl Zeiss) for acquisition of high resolution time series imaging. Fluo-4 AM was illuminated at 488nm and emission was recorded between 490 and 560 nm. Time series images were acquired at single cell resolution of 80x magnification, every 17 s. Up to 10 wells could be imaged simultaneously. Progression of glucose challenges and time of the stimulation during imaging was as follows. Imaging started with 5 min incubation in low glucose Krb containing 2mM glucose. This was followed by a 5 min incubation in high glucose Krb containing 20mM glucose. Sequential 5 min low and high glucose challenges were repeated two more times. Finally, wells were incubated in Krb containing 30mM KCl as a depolarization challenge. Between the stimulations, imaging was stopped and clusters were quickly washed with 2mM glucose Krb buffer. Then the next low or high glucose solution was added and imaging resumed. Fluorescence intensity changes during imaging were analyzed using ImageJ /Fiji by applying StackReg to correct for the movement of the clusters over the course of the imaging, and ROI manager to measure the fluorescence intensity of the cluster or cells within the cluster throughout the imaging. Analysis of the cluster was done by the measurement of the average fluorescence intensity of the whole cluster which we call population analysis or average fluorescence intensity of the individual cells within the cluster which we call single cell analysis.

Immunohistochemistry Differentiated cell clusters or islets were fixed by immersion in 4% PFA for 1 hr at room temperature (RT). Samples were washed 3 times with PBS, embedded in Histogel (Thermo), and sectioned at 10 μm for histological analysis. Sections were subjected to deparrafinization using Histoclear (Thermoscientific; C78-2-G) and rehydrated. For antigen retrieval slides were emerged in 0.1M EDTA (Ambion; AM9261) and placed in a pressure cooker (Proteogenix; 2100 Retriever) for two hours. Slides were blocked with PBS + 0.1% Triton X-100 (VWR; EM-9400) + 5% donkey serum (Jackson Immunoresearch; 017-000-121) for 1 hr at RT, followed by incubation in blocking solution with primary antibodies overnight at 4°C. The following primary antibodies were used 1:100 unless otherwise noted: goat anti-human PDX-1/IPF1 (R&D Systems; AF2419), mouse anti-Nkx6.1 (University of Iowa, Developmental Hybridoma Bank; F55A12-supernatant) (1:100), rat anti-insulin (pro-)/C-peptide (Developmental Studies Hybridoma Bank; GN-ID4), mouse anti-glucagon (Abcam; ab82270), goat anti-somatostatin (Santa Cruz Biotechnology sc7819), and guinea pig anti-insulin (Dako; A0564). Cells were washed twice in PBS the next day, followed by secondary antibody incubation for 2 hr at RT (protected from light). Secondary antibodies conjugated to Alexa Fluor 488 or 594 were used to visualize primary antibodies. Following two washes with PBS, the histology slides were mounted in Vectashield mounting medium with DAPI (Vector Laboratories; H-1200), covered with coverslips and sealed with nail polish. Representative images were taken using an Olympus IX51 Microscope or Zeiss LSM 510 or 710 confocal microscope.

Flow Cytometry Differentiated cell clusters or islets were dispersed into single-cell suspension by incubation in TrypLE Express (Invitrogen) at 37°C until clusters dissociated to single cells upon mixing by pipetting gently up and down (typically 10-15 min). The TrypLE was quenched with 3-4 volumes of culture media and cells were spun down for 3 min at 1000rpm. Cells, generally 1-2 million, were washed once in PBS (1mL) and transferred to a 1.7ml microcentrifuge tube (Bioscience; 11510). Cells were resuspended in 4% PFA and incubated on ice for 30 min. Cells were then washed once in PBS followed by incubation in blocking buffer (PBS + 0.1% Triton X-100 + 5% donkey serum) on ice for 1 hr. Cells were then resuspended in blocking buffer with primary antibodies and incubated at 4°C overnight. Primary antibodies, diluted 1:300 unless otherwise noted: Mouse anti-Nkx6.1, rat anti-insulin (pro-)/C-peptide, mouse anti-glucagon, and goat anti-somatostatin. Cells were washed twice in blocking buffer and then incubated in blocking buffer with secondary antibodies on ice for 2 hr (protected from light). Secondary antibodies conjugated to Alexa Fluor 488 or 647 (Life Technologies) were used to visualize primary antibodies. Cells were then washed 3 times in sorting buffer (PBS + 0.5% BSA (Sigma; A8412) and finally resuspended in 500-700μl sorting buffer, filtered through a 40μm nylon mash into flow cytometry tubes (BD Falcon; 352235), and analyzed using the LSR-II flow cytometer (BD Biosciences) with at least 30,000 events recorded. Analysis of the results was performed using FlowJo software.

Gene Expression Analysis of Sorted Cells Hrvatin et al., 2014 Hrvatin S.

O’Donnell C.W.

Deng F.

Millman J.R.

Pagliuca F.W.

DiIorio P.

Rezania A.

Gifford D.K.

Melton D.A. Differentiated human stem cells resemble fetal, not adult, β cells. To analyze global gene expression of SC-β cells, we used a recently described fixation and sorting strategy to isolate NKX6-1+/INS+ SC-β cells from the heterogenous cell clusters (). Two independent differentiation batches of SC-β clusters were harvested in single cell suspension using TrypLE and fixed in 4% PFA containing RNasin (VWR PAN2615) on ice for 30 min. Fixed cells were incubated with primary antibodies (mouse anti-NKX6-1 diluted 1:100 and guinea pig anti-insulin diluted 1:100) for 30 min in buffer containing RNasin, washed twice and then incubated with secondary antibodies (anti-mouse Alexa Fluor 488 and anti-guinea pig Alexa Fluor 647) in buffer containing RNasin for 30 min each. Hrvatin et al., 2014 Hrvatin S.

O’Donnell C.W.

Deng F.

Millman J.R.

Pagliuca F.W.

DiIorio P.

Rezania A.

Gifford D.K.

Melton D.A. Differentiated human stem cells resemble fetal, not adult, β cells. After antibody staining, cells were sorted by fluorescence activated cell sorting (FACS) to obtain at least 100,000 cells per sample. Samples were subsequently incubated in Digestion Buffer (RecoverAll Total Nucleic Acid Isolation Kit, Ambion AM1975) at 50°C for 3 hr, prior to RNA isolation according to manufacturer’s instructions. RNA concentration was quantified using Nanodrop 1000. Double-stranded cDNA was generated by reverse transcription from at least 100ng of total RNA according to manufacturer’s instructions (Illumina TotalPrep RNA Amplification Kit, Life Technologies, AMIL1791). At least 750ng cRNA per sample was hybridized to Human HT-12 Expression BeadChips (Illumina) using the Whole-Genome Expression Direct Hybridization kit (Illumina). Chips were scanned on the Illumina Beadstation 500. Raw data was adjusted by background subtraction and rank-invariant normalization (GenomeStudio software, Illumina). Before calculating fold change, an offset of 20 was added to all probe set means to eliminate negative signals. The p-values for differences between mean signals were calculated in GenomeStudio by t test and corrected for multiple hypotheses testing by the Benjamini-Hochberg method in combination with the Illumina custom false discovery rate (FDR) model. These SC-β cell microarray data and the previously published hPSC, PH, fetal β and adult β cell data () were imported into the R statistical computing platform using the programming packages lumi and EMA. Samples were analyzed by hierarchial clustering using Pearson’s correlation and Ward linkage. The pattern of clustering was robust to other distance and linkage metrics. Raw microarray data will be uploaded to publicly available databases.

Electron Microscopy To analyze granular ultrastructure, cadaveric pancreatic islets or differentiated clusters were fixed at room temperature for 2 hr with a mixture containing 1.25% PFA, 2.5% glutaraldehyde, and 0.03% picric acid in 0.1 M sodium cocodylate buffer (pH 7.4). Samples were then washed in 0.1M cacodylate buffer and postfixed at room temperature for at least 2 hr with a mixture of 1% Osmium tetroxide (OsO4) and 1.5% Potassium ferrocyanide (KFeCN6). Next samples were washed in 0.1M cacodylate buffer and postfixed with 1% Osmiumtetroxide (OsO4)/1.5% Potassiumferrocyanide (KFeCN6) for 1 hr. After washing three times with water, samples were stained in 1% aqueous uranyl acetate for 1 hr, washed twice in water and subsequently dehydrated in grades of alcohol (10min each; 50%, 70%, 90%, 2x10min 100%). A one hour incubation in propyleneoxide was followed by infiltration overnight in a 1:1 mixture of propyleneoxide and TAAB Epon (Marivac Canada, St. Laurent, Canada). After the overnight infiltration, TAAB Epon was used to embed the samples and they were polymerized at 60°C for 48 hr. Sample sections were cut ultrathin (approximately 60nm) on a Reichert Ultracut-S microtome and placed onto copper grids. These were then stained with 0.2% lead citrate. A JEOL 1200EX Transmission electron microscope or a TecnaiG2 Spirit BioTWIN was used to analyze the samples. Images from at least two independent pancreatic donors and at least two independent different batches for each protocol were recorded with an AMT 2k CCD camera. ImageJ software was used to analyze and quantify images. For determination of granular protein composition, immunogold labeling was performed on cadaveric pancreatic islets or differentiated clusters. Samples were washed with PBS and then placed into 0.5mM EDTA in PBS. A 200μl cushion of 8% paraformaldehyde (in 0.1M Sodium Phosphate buffer, pH 7.4) was then layered with 800 μl of sample. The samples were pelleted for 3 min at 3000 rpm then placed into 4% paraformaldehyde for 2h at room temperature. Cell pellets were then washed with PBS and treated with 2.3M sucrose and 0.2M glycin in PBS for 15 min, then placed into liquid nitrogen prior to being sectioned at −120°C. Sections were then transferred to formvar-carbon coated copper grids were stored at 4°C in PBS or 2% gelatin until immunogold labeling was performed. The gold labeling process was performed at room temperature on a piece of parafilm. The following primary antibodies were used: 1:500 mouse anti-glucagon (Abcam; ab82270) and guinea pig anti-insulin (Dako; A0564). Primary antibody and protein-A gold were first diluted in 1% BSA in PBS, before staining. The diluted antibody solution was centrifuged for 1 min at 14000 rpm. Sample grids were blocked with 1% BSA for 10 min then incubated in 5μl drops of primary antibody solution for 30 min. The grids were then washed in 4 drops of PBS for 15 min, then incubated with 5μl drops of 5nm or 15nm protein-A gold for 20 min, labeling insulin and glucagon, respectively. Protein-A labeled samples were then washed in 4 drops of PBS for 15 min and then 6 drops of double distilled water. Labeled grids were then contrasted and embedded in 0.3% uranyl acetete in 2% methyl cellulose for 10 min. Grids were picked up with metal loops then examined in a JEOL 1200EX Trans or a TecnaiG2 Spirit BioTWIN mission electron microscope. Images from at least two independent pancreatic donors and at least two independent different batches were recorded with an AMT 2k CCD camera.

SCID-Beige Transplantation Studies Immunodeficient SCID-Beige mice, aged 8-10 weeks, were procured from Taconic or The Jackson Laboratory. Human islets (500-1000 IEQ per animal; Prodo Laboratories) or hPSC derived cell clusters (5 million cells per animal) were resuspended in 200ul RPMI1640 media (Life technologies; 11875-093). Cells were aliquoted into PCR tubes and kept on ice for 5 to 10 min before the loading into a catheter for cell delivery below the mouse kidney capsule. To prepare each catheter, an infusion set 23G × ¾” (Terumo; SB∗S23BL) connected to a 1mL syringe was rinsed with 1ml of RPMI1640 media supplemented with 5% FBS serum (Corning; 35-011-CV). The catheter was then loaded with 0.6 ml of RPMI1640 media (no serum added). Cell clusters were loaded through the tip of the catheter needle and placed vertically to settle to the bottom of the catheter tubing for 5 min in room temperature. During the catheter cell loading step, mice were anesthetized with avertin 1.25% (250mg/kg; 0.5ml/25 g 1.25% Avertin/body weight), and the left ventricle surgical site was shaved and disinfected with betadine and alcohol. An incision of approximately 1 cm was made to expose the kidney. The catheter needle was inserted directly under the kidney capsule and cells injected. The abdominal cavity was closed with PDS absorbable sutures (POLY-DOX; 2016-06) and the skin was closed with surgical clips (Kent Scientific Corp; INS750346-2). Mice were placed on a micro-temp circulating pump and blanket (∼37°C) during the surgery/recovery period to aid in the rapid recovery of mice following anesthesia and given a dose of 5 mg/mkg carprofen right after the surgery and 24 hr after the initial dose. Wound clips were removed 14 days after recovery from surgery and the mice were monitored twice a week. After two weeks of recovery from surgery, animals were analyzed by performing a glucose challenge and collecting serum for human insulin concentration measurement. After fasting the mice for 16 hr overnight, the glucose challenge was performed by intraperitoneal (IP) injection of 2g D-(+)-glucose/1 kg body weight and blood was collected at 30 min post glucose injection through facial vein puncture using a lancet (Feather; 2017-01). For a subset of animals, serum was collected both preinjection and 30 min postinjection in order to measure glucose responsiveness of the human insulin producing cells in vivo. Serum was separated out using Microvettes (Sarstedt 16.443.100) and stored at −80 C until ELISA analysis. Serum human insulin levels were quantified using the Human Ultrasensitive Insulin ELISA. Kidneys containing the grafts were dissected from the mice, fixed in 4% PFA overnight, embedded in paraffin, and sectioned for histological analysis. Immunohistochemistry analysis was performed as described above. All animal experiments were performed in accordance with Harvard University International Animal Care and Use Committee (IACUC) regulations.

NRG-Akita Transplantation Studies tm1Mom IL2rgtm1Wjl Ins2Akita (NRG Akita) ages 8-12 weeks were generously provided by Dr. Leonard D. Shultz of The Jackson Laboratory, Bar Harbor, ME. This stock is maintained by mating NOD-Rag1null IL2rγnull Ins2+/Akita males with NOD-Rag1null IL2rγnull females and previous studies have determined that transplantation of 4000 IEQ human islets restores normoglycemia in these mice ( Brehm et al., 2010 Brehm M.A.

Bortell R.

Diiorio P.

Leif J.

Laning J.

Cuthbert A.

Yang C.

Herlihy M.

Burzenski L.

Gott B.

et al. Human immune system development and rejection of human islet allografts in spontaneously diabetic NOD-Rag1null IL2rgammanull Ins2Akita mice. null IL2rγnull Ins2+/Akita. Development of spontaneous diabetes was evaluated in cohorts of nonfasted NRG-Akita mice by blood glucose measurements with an ACCUCHEK active glucometer (Hoffman-LaRoche, Basel, Switzerland). Mice were housed in a specific pathogen free facility in microisolator cages. Blood glucose levels were determined in nonfasted mice prior to their entry into the experiment for confirmation of hyperglycemia prior to cell transplantation. For transplantation, the mice were fasted for 16 hr, fasted blood glucose levels were determined, and mice were transplanted with five million SC-β cells or control PH cells. The cells were aliquoted into individual sterile 1.5 ml eppendorf tubes, allowed to settle for 15 min, and the supernatant was removed using a sterile pipette. Ten microliters of NRG whole blood was then added to each cell pellet and allowed to form a clot with the cells. NOD.Cg-Rag1IL2rgIns2(NRG Akita) ages 8-12 weeks were generously provided by Dr. Leonard D. Shultz of The Jackson Laboratory, Bar Harbor, ME. This stock is maintained by mating NOD-Rag1IL2rγIns2males with NOD-Rag1IL2rγfemales and previous studies have determined that transplantation of 4000 IEQ human islets restores normoglycemia in these mice (). All NRG-Akita mice used in the experiments were NOD-Rag1IL2rγIns2. Development of spontaneous diabetes was evaluated in cohorts of nonfasted NRG-Akita mice by blood glucose measurements with an ACCUCHEK active glucometer (Hoffman-LaRoche, Basel, Switzerland). Mice were housed in a specific pathogen free facility in microisolator cages. Blood glucose levels were determined in nonfasted mice prior to their entry into the experiment for confirmation of hyperglycemia prior to cell transplantation. For transplantation, the mice were fasted for 16 hr, fasted blood glucose levels were determined, and mice were transplanted with five million SC-β cells or control PH cells. The cells were aliquoted into individual sterile 1.5 ml eppendorf tubes, allowed to settle for 15 min, and the supernatant was removed using a sterile pipette. Ten microliters of NRG whole blood was then added to each cell pellet and allowed to form a clot with the cells. The recipient mice were anesthetized by intraperitoneal injection of 5 μl/gram of a mixture of 15 mg/ml ketamine and 2 mg/ml xylazine using a 1 ml syringe with a 25 gauge needle. Upon induction of anesthesia, the hair at the surgical site was removed using electrical clippers and the loose hair removed using gauze slightly dampened with ethanol. The skin area was cleaned with 0.1% topical solution of betadine followed by a 70% ethanol solution, and this cleaning was repeated three times. All surgery was performed using aseptic technique in a sterile hood under aseptic conditions using sterilized gauze pads as surgical draping. An incision approximately 2.0 cm was made through the skin and then through the underlying musculature and the kidney gently pulled through the incision space. A 16 gauge trocar was used to implant the blood clot containing the cells under the subrenal capsule and the graft bearing kidney was placed back into the peritoneal cavity. A 4-0 coated vicryl suture (Ethicon vicryl J464G) using an interrupted stitch was used to suture the musculature and the skin site was then closed using surgical wound clips (BD 427631). Animals were kept warm during recovery from surgery and were given a single i.p. dose of 2.4mg/kg buprenorphine SR-LAB to provide pain relief for 72 hr (ZooPharm BZ8069317). The mice were monitored three times a week and fourteen days later, the surgical wound clips were removed. At select times, the animals were fasted for 16 hr, a baseline blood glucose was determined and the mice were challenged with an intraperitoneal injection of 2 g/kg D-(+)-glucose. Thirty minutes post glucose challenge blood glucose levels were determined and ∼150 μl of blood was obtained through facial vein puncture using a lancet, allowed to clot for 60 min, and serum recovered and stored at −80°C until levels of human insulin were determined by ELISA. All animal use was in accordance with the guidelines of the Institutional Animal Care and Use Committees of the University of Massachusetts Medical School and The Jackson Laboratory in conformity with the recommendations in the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, National Research Council, National Academy of Sciences, 1996).