Human ES cell growth and differentiation to pancreatic beta-like cells
Highly pluripotent, clinical grade, human ES cells HADC-100 (12), (provided by Professor Benjamin Reubinoff, Hadassah Medical School, Jerusalem, Israel) were grown to confluent monolayers in essential E8 medium (Gibco, Cat#A1517001), with addition of penicillin and streptomycin (PS, Gibco, Cat#15140-122) on vitronectin-coated flasks (Gibco, Cat# A14700). Differentiation was performed on cell aggregates formed in spinner flasks during two days in dynamic suspension cultures. In brief, 48 hours before starting the differentiation protocol (day-2), non-differentiated cells were dissociated with Versene (Gibco, Cat#15040033). Single cells washed with PBS−/− (Gibco, Cat#14190-094) were seeded in 500 ml disposable spinner flasks (Corning, Cat#CZ-3153), filled with 250 ml E8 medium containing 10µM Rock Inhibitor Y27632 (Cayman Chemical, #10005583-10), at concentration of 0.8-1 × 106 cells/ml. The spinner flasks were placed on an orbital shaker (Senova) at speed of 70 rpm in a humidified incubator set at 5% CO2 and 37 °C. This resulted in the formation of ES cell clusters in suspension, as well as in cell proliferation. On day − 1, 80% of the E8 medium was replaced. On Day 0, the E8 medium was washed away (13) by letting the aggregates settle for 5 minutes and removing the supernatant with a pipette. Cells were washed with 250 ml PBS−/−; after 3 min stirring in the incubator, PBS−/− was replaced by 250 ml of stage1 differentiation medium. The media for the seven-stage differentiation protocol, refined on the basis of several published protocols (4, 5, 14–17), are detailed in additional files 1 and 2 (Table S1 and Table S2).
For Bioreactors, the hESC aggregation and differentiation was similarly done in the DASbox mini (Eppendorf) with online monitoring of culture parameters. Four parallel bottles containing 100–150 ml medium were seeded with 0.8-1 × 106 cells/ml 48 h before and washed with PBS just before differentiation as above. Medium changes were done batch-wise in the semi-closed system using peristaltic pumps. The differentiation protocol was the same as for spinner flasks.
Real time quantitative PCR
RNA was isolated from cells using the RNAeasy micro kit (Qiagen #74004) and purified from genomic DNA with RNase-free DNase kit (Qiagen #79254). The cDNA synthesis was done with the high capacity cDNA Reverse Transcription kit (Applied Biosystems 4368814). Transcript levels were measured by real-time qPCR using Taqman Fast advanced master mix (Applied Biosystems #4444557). The level of each gene was normalized to endogenous HPRT gene, using the 2−ΔΔCT method. The probes used for qPCR are listed in Table S3. The MARIS procedure (18) is described in Additional files.
Flow cytometry.
Samples from settled aggregates during or at the end of the differentiation process were dissociated with Accumax (Sigma, Cat# A7089) (0.35 ml per 300,000 estimated cells) for 8–10 minutes, after which the enzyme was blocked in 1 ml PBS−/− with 10% FBS, and the cells were centrifuged (350xg, 3 min). For external cell membrane labelling, cells were washed in PBS−/−, and antibodies (e.g. anti-CD49A, as listed in supplementary Table S4) were directly added to the cell suspension in FACS buffer (0.5% BSA in PBS) followed by incubation at 4oC for 30 min. For internal antigen labelling (e.g. anti-human C-peptide and Nkx6.1, Table S4), cells after centrifugation were washed once in 1 ml PBS and fixed in 0.4 ml of 4% paraformaldehyde (PFA, EMS Cat# 15710), for 20 min at 4oC. After two washes with PBS cells were incubated for 1 h at 4oC in blocking solution (PBS with 5% Bovine serum albumin (BSA) and 3% horse serum) containing 0.4% Triton x-100 (Sigma, T6878), and washed once with PBS. The cell pellet suspended in 0.1 ml blocking solution (with 0.1% Triton x-100) containing antibodies, was incubated for 14–16 hours at 4oC, or for 1 h at room temperature (RT), and washed with PBS. When fluorescent tag-conjugated primary antibodies were used, fluorescence was read after this step in Flow Cytometer-BD FACS Canto II. Otherwise, cells were incubated in blocking solution containing 1:100 dilutions of the fluorescent tag-conjugated secondary antibody and washed with PBS before reading fluorescence.
Functional cell capture screening (FCCS) on antibody arrays.
Antibody arrays were printed in a Microgrid printer with solid pins (Total array Systems, BioRobotics, Cambridge, UK) on hydrogel-coated slides (Full Moon Biosystems, Sunnyvale, CA) using a panel of 235 monoclonal mouse anti-human antibodies (BD biosciences), each antibody being spotted at five different places in the array, as described before (10, 11) (US patent 2018 / 0369290 A1, Item 0081). The cell clusters were dissociated using TrypLE Express (Invitrogen Cat#12604) for 4 min, followed by quenching with 10% FBS in PBS, and centrifugation and resuspension in CMRL. The printed area of the array was blocked for 3 minutes with 1% BSA in PBS solution, before cell seeding, at about 0. 5 × 10 6cells/ml in 0.25–0.5 ml of CMRL medium, supplemented with 2 µl of DNase I (Ambion 2U/µl) and incubation was for 1 hour at 37oC. Excess cells were removed in a large volume of PBS and the arrays were fixed in 4% (w/v) paraformaldehyde for 10 minutes. Cells on the array were permeabilized in PBS, 0.2% (v/v) Triton x-100 for 20 min, washed twice with PBS and blocked for 45 min in blocking buffer (2% FBS, 2% BSA, 50 mM glycine in PBS). After blocking, arrays were washed twice with PBS and incubated for 2 hours at RT in blocking buffer with 0.1% of Triton x-100 containing the primary antibody guinea-pig anti-insulin (DAKO, A0564). Primary antibodies were removed, and arrays were washed three times with working buffer. Then, secondary antibodies were added in working buffer for 45 min at room temp: cy5 donkey anti-guinea–pig, (Jackson ImmunoResearch 706 − 175 -148). Arrays were washed three times in working buffer and imaged using automated, high content fluorescence microscopy (IXmicro, MDC ). Total cells in each spot were counted by phase microscopy and the percent of insulin positive cells was calculated. Three repeats were performed with different batches of ES-derived cells at stage 7 of the differentiation protocol. The significance of the amount of cell binding to surface antibody was evaluated by two sample paired T-test (p value less than 0.05). In another set of experiments, cells were reacted with antibodies against insulin as above but also with antibodies against Pdx1 and against Nkx6.1 (see Table S4). The number of cells stained for PDX1 and Insulin, and for insulin and Nkx6.1 was counted.
Magnetic Activated Cell Sorting (MACS)
Cell clusters after stage 7 of differentiation were washed in PBS−/−,dissociated with Accumax (10 ml for 25 × 106 cells) for 10 min at 37oC,, washed with CMRL 2% BSA and filtered through a 30 µm MACS filter previously washed with PBS−/−, to eliminate cell aggregates, and counted using Nucleocounter® NC-200. Part of the clusters was set aside for control (non-dissociated, non-selected cells), seeded at 106 cells per ml in ultra-low binding 6-well-plates (Corning Cat#CLS3471) and left in the incubator on a stirrer (Dura Mag 9 position digital stirrer, CHEMCELL) set at 95 rpm in the 37oC incubator for three days in medium CMRL+ (Table 2s), before implantation. Cells are suspended in MACS buffer (PBS−/−, 2% BSA, 2 mM EDTA, sterile, degassed (100 µl per 107 cells), for reaction with anti-CD49A (Myltenyi, cat# 130-101-397) using 10 µl per 107 cells for 10 min at 4oC, followed by washing with 5 ml MACS buffer. Cells suspended in MACS buffer (80 µl per 107 cells) were reacted with 20 µl per 107 cells of anti-PE microbeads (Miltenyi; cat# 5181214192) for 15 min at 4oC. After washing with 5 ml cold MACS buffer and centrifugation, cells were suspended in MACS buffer and applied to pre-separation filters and LS MACS column(s) as recommended by the manufacturer. Prior to implantation, all single cells fractions were re-aggregated in suspension in non-TC treated 6-well plates (Corning Cat#CLS3471-24EA) in CMRL +, 10 µM RI Y27632 and 2µ g/ml Laminin (Bio Lamina Cat#MX521CTG), on the stirrer, set at 95 rpm.
Table 2
Proportions of C-peptide and Nkx6.1 subpopulations in ILC cells double selected with antibodies to CD26 and to CD49A
Expt 1 |
Cell fraction sorted | Total Non sorted | CD26 enriched | CD26 depleted | CD26 depleted CD49A enriched | CD26 depleted CD49A depleted |
Cell markers: | Percent of cells |
1. C-peptide/NKX6.1 DP | 24.2 | 19.7 | 33.8 | 71.5 | 16 |
2. C-peptide only | 22.9 | 43.0 | 4.8 | 10.7 | 4.0 |
3. NKX6.1 only | 35.8 | 18.0 | 52.8 | 12.1 | 69.5 |
4. Negative | 17.1 | 19.3 | 8.5 | 5.7 | 10.5 |
Expt 2 |
Cell fraction sorted | Total | Total Reaggregated | CD26 enriched | CD26 depleted | CD26 depleted CD49A enriched | CD26 depleted CD49A depleted |
Cell markers | Percent of cells |
1. C-peptide/NKX6.1 DP | 22.5 | 32.6 | 6.1 | 33.7 | 66.6 | 21.2 |
2. C-peptide only | 20.2 | 21.7 | 43.8 | 8.4 | 11.4 | 7.0 |
3. NKX6.1 only | 13.3 | 21.6 | 6 | 24.3 | 13.3 | 42.7 |
4. Negative | 44.1 | 24.2 | 44.1 | 33.6 | 8.7 | 29.1 |
The C-peptide/Nkx6.1 DP cells predominate in CD26 depleted/CD49 enriched fraction. Quantitation of double MACS sorting as described in Fig. 5. Two independent fractionation experiments are shown. |
For removal of CD26 positive cells prior to CD49A enrichment, the cells were dissociated as above, incubated for 10 minutes at 4oC with anti-CD26-PE (cat#302706), (10 ul/ 107 cells, in 100 ul MACS buffer for 107 cells), and after washing, reacted with anti PE antibody as described above. The mixture, after washing and resuspension in MACS buffer, was applied to LS column(s) and the flow through fraction (CD26 depleted) kept for further fractionation by MACS with anti-CD49A antibody.
Diabetes induction in mice and ILC implantation.
Immuno-competent mice C57BL/6JOlaHsd (Harlan, Israel), 7-week-old males or female, were rendered diabetic by intraperitoneal injection of streptozotocin (STZ) (Sigma, Cat#S0130). As indicated, doses were either 150 mg STZ/kg, in one single injection, or 4 daily injections of 50 mg STZ/kg, after 6 hours fasting. Implantation was performed in diabetic mice, defined by blood glucose higher than 200 mg/dL for three consecutive days. Blood glucose was measured by a glucometer, on tail vein blood. Intraperitoneal Glucose Tolerance Test (IPGTT) was done after fasting the mice overnight by IP. injection of glucose (2 g/kg). Blood glucose was then monitored during a 2 hour period.
For implantation of micro-encapsulated ILC cells (see below), mice were anesthetized by an IP injection of ketamine/xylazine (Sigma, K4138) 85%/15% ratio (v/v) and then mounted on a surgical pad. The skin was prepared by shaving with electric clippers, application of Polydin, and then 70% ethanol solution. An abdominal incision (1 cm), and peritoneal incision (1 cm) allowed to insert microencapsulated ILCs into the peritoneal cavity of the mouse using a 1 ml sterile plastic tip (about 0.5 ml total volume). The peritoneum and the skin were closed with sutures and cleaned with Polydin. The mice were kept warm by a heating pad till they woke up. The cell doses implanted were between 1.0 and 2.5 × 106 cells, as indicated.
Preparation of TMTD-modified alginate and microcapsules.
Triazole thiomorpholine dioxide (TMTD Y1-Z15) preparation and coupling to alginate PRONOVA UP-MVG alginate (NovaMatrix) were done for Kadimastem, by Recipharm-Israel as described [8, 9]. After verification of the product structure by NMR, purification by filtration, dialysis and desiccation, elemental analysis revealed that more than 50% alginate guluronic or mannuronic residues were coupled to TMTD. Solutions of 4.6% of TMTD-coupled alginate were used (80% in volume of 5% (w/v) TMTD-coupled UP MVG and 20% of 3% (w/v) UP MVG). Stage 7 ILCs (see Table S1), washed with KREBS buffer without Ca++ pH 7.4, were mixed with alginate solutions at the concentration of about 107 cells /ml in a 5 ml Eppendorf tube. The micro-encapsulator Buchi B395, located in a tissue culture laminar flow hood for sterility, was set up to obtain microcapsules of 1.5 mm diameter with 4.6% alginate polymerized in CaCl2 (100 mM in HEPES pH 7.4).
ILC Immunostaining and Imaging.
Stage 7 cell clusters fixed in 4% PFA and washed in PBS−/−. A minimum volume of liquid 1% agarose was added to the pellet of clusters. After agarose became solid, the block was embedded in paraffin and 10 µm thick sections were produced and bound to glass slides. After removal of paraffin by alternative baths of xylene and ethanol, antigen retrieval was performed by heating slides in 10 mM citrate buffer pH 6.0 (ZYTOMED systems), by heating for 15 min in pressure cooker (Bio TintoRetriever). Blocking nonspecific binding and cell permeabilization was done by incubation with PBS containing 5% BSA, 3% horse serum and 0.3% Triton x-100 (blocking solution) 500 ul added per slide and slides covered with parafilm for 1 h at RT. Antibodies against PDX1, C-peptide, Nkx6.1 (Table S4) were diluted in blocking solution and incubation done overnight at 4oC in humidified chambers. After two washes in PBS, incubation with secondary antibodies was done for 1hr at room temperature and washes were done similarly. Nuclei were stained with DAPI (1µ g/ml). The slides were mounted with aqueous mounting medium (2 drops) and covered with coverslip. Images were obtained using Nikon Eclipse 80.i fluorescence microscope and a DS.fi1 camera combined with NIS Elements computer program.