Culture of ESCs
Mouse ESCs (ES-E14TG2a) from 129/Ola mice were purchased from the American Type Culture Collection (ATCC). ESCs were maintained in high-glucose Dulbecco’s Modified Eagle Medium (DMEM; Gibco BRL, USA) with 10% fetal bovine serum (FBS; Gibco BRL, USA), 10 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), 0.12% sodium bicarbonate (NaHCO3), 0.1 mM nonessential amino acids, 0.1 mM β-mercapto-ethanol, 100 U/mL penicillin, 100 µg/mL streptomycin, and 1000 U/mL leukemia inhibitory factor (LIF) at 37 °C in an environment with 5% CO2. The medium was refreshed once daily, and cells were passaged once every 2–3 days.
Culture of embryoid body (EB)
The EB culture medium was based on ESCs culture medium except for LIF and contained an additional 25 ng/mL Wnt3a and 50 ng/mL Activin A[17].
Tet-On system
The Tet-On system was used to construct the Pdx1 over-expressing lentivirus vector with green fluorescent protein (GFP) and puromycin resistance. The Pdx1 recombinant plasmid was constructed and then transfected into 293T cells, and fluorescence microscopy was done to observe the fluorescent marker (GFP). Western blotting was done to detect Pdx1 protein expression. The recombinant lentivirus plasmid and two original vector plasmids were co-transfected into 293T cells with Lipofectamine 2000 (Invitrogen, USA) according to the manufacturer’s instructions. At 8 h after transfection, the medium was refreshed with complete medium, and the supernatant was harvested 48 h later. The supernatant was then concentrated to obtain the lentivirus concentrate. The viral titer was determined in 293T cells.
Transfection of ESCs with lentivirus
Cells were divided into the blank control group (ESC), the blank vector group (Pdx1− ESC), and the Pdx1 lentiviral vector group (Pdx1+ ESC). ESCs were seeded into 6-well plate. Sixteen h later, confluence reached 20–30%, and then transfection was done. In brief, the medium was removed, and serum-free medium (894 µL) was added. Then, 100 µL of Polybrene (concentration: 5 µg/mL) and 6 µL of viral solution (titer: 1E + 9TU/mL) were added at a final volume of 1 mL. After 12-h transfection, the medium was refreshed with 2 mL of medium containing 10% FBS. The medium was refreshed once daily. After 48-h transfection, cells were passaged at a ratio of 1:5 when confluence reached > 80%. At 24 h after passaging, puromycin was added at a final concentration of 800 ng/mL (total volume of the medium: 2 mL). After puromycin screening for 2 days, the medium was refreshed with puromycin-free medium.
Detection of transfection efficiency by flow cytometry and construction of ESCs with stable Pdx1 expression and negative control ESCs
Cells in the Pdx1− ESC group and the Pdx1+ ESC group were screened with 800 ng/mL puromycin for 2 days and induced with 2 µg/mL doxycycline (DOX). Then, cells were harvested from the ESC group, the Pdx1− ESC group, and the Pdx1+ ESC group, and washed in phosphate-buffered saline (PBS) once. 1.0 × 106 cells were resuspended with 200 µL of PBS and then loaded for the detection of transfection efficiency by flow cytometry. The other cells were resuspended with PBS at a density of 1 × 107 cells/mL. Flow cytometry was used to sort GFP+ cells in the Pdx1− ESC group and the Pdx1+ ESC group, and cells in the ESC group served as controls. After sorting, positive cells were seeded into 6-well plates, followed by incubation at 37 °C in an environment with 5% CO2 (d0). On the second day (d1), puromycin was added at a final concentration of 800 ng/mL. On day 3 (d3), puromycin-resistant colonies could be observed. Cells surrounding the colonies were digested with trypsin, and the cell colonies were collected into 96-well plates for further culture. Twenty-four later, puromycin was added at a final concentration of 400 ng/mL to maintain the colonies. Then, cells were sequentially transferred into 24-well plates, 12-well plates, and 6-well plates for culture and expansion, and cell lines with stable transfection were harvested.
Observation of Pdx1 gene expression under fluorescence microscope
Cells of the three groups were seeded into 6-well plates (2 wells/group). In one well for each group, DOX was added at a final concentration of 2 µg/mL 24 h later. Cells were then observed under the fluorescence microscope.
Cell cycle and apoptosis of ESCs before and after transfection
Cells of the three groups were seeded into 6-well plates. Cells were cultured with ESC medium followed by incubation at 37 °C in an environment with 5% CO2. They were harvested and washed in PBS 48 h later. Cells were resuspended in 2 mL of PBS. In total, 2–5 × 105 cells were resuspended in 400 µL of binding buffer containing Ca2+ after centrifugation. The cell suspension was divided into two tubes (200 µL of suspension in each tube). In one tube, cells were not treated and served as blank controls; in the other tube, FITC-conjugated Annexin V (5 µL) was added, followed by incubation at room temperature for 15 min in the dark. Then, 10 µL of propidium iodide (PI) was added, and cells were subjected to flow cytometry.
Cells were collected and single-cell suspensions were prepared with 200 µL of PBS after centrifugation. Cells were then fixed in 2 mL of 75% ice-cold ethanol (absolute ethanol: water = 7:3; − 20 °C) at 4 °C overnight. After washing in PBS once, cells were treated with 50 µL of RNAse and 450 µL of PI staining solution, followed by detection of cell cycles in 4 h.
Sorting of CXCR4+ definitive endodermal cells with immune beads
Cells of the three groups were maintained for 5 days until a high proportion of DE cells at the EB stage was present after differentiation. Cells were collected and resuspended in PBS after centrifugation. Cells were then treated with phycoerythrin (PE)-conjugated anti–mouse CXCR4 monoclonal antibody (10 µL/1.0 × 106 cells) at 4 °C for 20 min. After washing in running buffer twice, cells were resuspended in running buffer (80 µL/1 × 107 cells), and anti–PE immune beads (Miltenyi Biotec) were added (20 µL/1 × 107 cells), followed by incubation at 4 °C for 15 min. After washing in running buffer twice and centrifuging, cells were resuspended in running buffer at a density of 1 × 107 cells/mL. Cells were then subjected to flow cytometry (FACSVerse) for sorting CXCR4+ cells. The data were collected with EXPO32 MultiCOMP ver. 1.1C software, and analyzed with EXPO32 analysis ver. 1.2B software.
Differentiation of CXCR4+ DE cells into pancreatic-like cells
After sorting, CXCR4+ DE cells in the ESC group, the Pdx1− ESC group, and the Pdx1+ ESC group were seeded into 6-well plates (0.5–1.0 × 105cells/well) on day 0. On the second day (d1), DOX was added at the final concentration of 2 µg/mL. The Tet-On system was used to initiate Pdx1 expression in DE cells. Cells in the three groups were cultured for 4 (d3), 8 (d7), 11 (d10), and 15 (d14) days.
Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)
Total RNA from samples was extracted using TRIzol® Reagent (Life Technologies, Carlsbad, CA, USA), and the concentration was calculated from the absorbance at 260 nm as determined using an ND-2000 instrument (NanoDrop Technologies, Wilmington, DE, USA), followed by reverse transcription with PrimeScript™ Master Mix (TaKaRa Bio, Kusatsu, Shiga, Japan). RT-qPCR was performed on a CFX Connect™ real-time PCR detection system (Bio-Rad, Hercules, CA, USA) using SYBR® Premix Ex Taq™ (TaKaRa Bio, Kusatsu, Shiga, Japan). The primers are shown in Table 1–3. Data were analyzed using the ΔΔCt method with β-actin as an internal control. The RT-qPCR experiments were performed in sextuplicate using independent samples.
Table 1
Gene | Primer |
mouse Pdx1 | Forwar: 5‘-GGCAGGAGGTGCTTACACAG-3’ Reverse: 5‘-GGCCGGGAGATGTATTTGTT-3’ |
mouse 18sRNA | Forward: 5‘-GCTAGGAATAATGGAATAGG-3’ Reverse: 5 ‘-ACTTTCGTTCTTGAGGAATG-3’ |
Table 2
Primer for Moiuse CK19,Ptfla,Pax6,Neuro D1,Insuline,Amylase
Gene | Primer |
Mouse CK19 | Forward: 5‘-AGGTCAGTGTGGAGGTGGA − 3’ Reverse: 5‘-CTCAATCCGAGCAAGGTAGG − 3’ |
Mouse Ptf1a | Forward: 5‘-AAACGGGCAGTAACCCTCTT − 3’ Reverse: 5‘-TCAGCAACAGCAGCAGAAGT − 3’ |
Mouse Pax6 | Forward: 5‘-ATCGGAGGGAGTAAGCCAAG − 3’ Reverse: 5‘-TAGCCAGGTTGCGAAGAACT − 3’ |
Mouse Neuro D1 | Forward: 5‘-TTGTCCCAGCCCACTACCAATTTG − 3’ Reverse: 5‘-TCGGCGGATGGTTCGTGTTTG − 3’ |
Mouse Insuline | Forward: 5‘-CGGAGACCCACAAAGAGAGA − 3’ |
| Reverse: 5‘-GGAGTCACAGCGAGAAGACC − 3’ |
Mouse Amylase | Forward: 5‘-ACTGCCAACAGCATAGCAAA − 3’ |
| Reverse: 5‘-TCAAACAGGTGGACAATAGCA − 3’ |
Mouse 18srRNA | Forward: 5‘-GCTAGGAATAATGGAATAGG-3’ Reverse: 5 ‘-ACTTTCGTTCTTGAGGAATG-3’ |
Table 3
Primer for Mouse Notch1,Notch2,Hes1,Hes5
Gene | Primer |
Mouse Notch1 | Forward: 5‘-CGTGGATGAGTGTGCTCTG − 3’ Reverse: 5‘-TCCCGTGTAGCCCTGTAGAC-3’ |
Mouse Notch2 | Forward: 5‘-GACTGGCGACTTCACTTTCG − 3’ Reverse: 5‘-CATCCACACAAACTCCTCCA-3’ |
Mouse Hes1 | Forward: 5‘-GTGGGTCCTAACGCAGTGTC − 3’ Reverse: 5‘-TCAGAAGAGAGAGGTGGGCTA − 3’ |
Mouse Hes5 | Forward: 5‘-TCCTCTGGATGTGGGAAGAC − 3’ Reverse: 5‘-CTAAGAATGACCCTCCTGCTG − 3’ |
Mouse 18srRNA | Forward: 5‘-GCTAGGAATAATGGAATAGG-3’ Reverse: 5 ‘-ACTTTCGTTCTTGAGGAATG-3’ |
Western blotting
The samples were incubated in RIPA buffer (Thermo Scientific, Carlsbad, CA, USA). And the protein concentration was determined using the bicinchoninic acid (BCA) protein assay kit (CW Biotech, China). Protein separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), and then transferred onto poly-vinylidene difluoride (PVDF) membranes. The membranes incubated with a 1:1000 dilution of Pdx1, CK19, Ptf1a, Pax6, Neuro D1, insulin, amylase, Notch1, Notch2, Hes1, or Hes5 (Abcam,Cambridge, UK)primary antibody at 4 °C overnight. After washing in TBST (tris-buffered saline with Tween 20), the membranes incubated with a 1:1000 dilution of secondary antibody, at room temperature, for one hour. The data were analyzed using relative intensity with β-actin as the internal control. The Western blot experiments were performed in three repetitions using independent samples.
Statistical analysis
Statistical analysis was performed with SPSS version 17.0. Quantitative data are expressed as mean ± standard deviation (mean ± SD). Comparisons were done using Student’s t test between groups and analysis of variance among groups, followed by Student’s t test with Bonferroni correction for comparisons of means between two groups. A value of p < 0.05 was considered statistically significant.