hESCs were cultured and passaged as our previous paper described . In brief, hESCs were cultured with the Irradiated CF1 feeder cells (3×104 cells/cm2) on the T25 flasks (Corning) coated with Matrigel (Becton–Dickinson). hESCs were maintained in DMEM/F12 (Invitrogen) supplemented with 20 % knockout serum replacement (Invitrogen), 4 ng/mL basic fibroblast growth factor (bFGF; Invitrogen), 2 mmol/L l-glutamine (Invitrogen), 1 % nonessential amino acids (Invitrogen) and 0.1 mmol/L β-mercaptoethanol (Sigma-Aldrich). hESCs were passaged approximately once a week. Collagenase IV was used to dissociate the cells from the feeders as cell clumps, which were dissociated to an appropriate size before being passaged onto newly prepared feeder cells.
TALENs efficiency detection
TALENs for CIITA were designed to target exon2 (2L1: gctgaccccctgtgcct; 2L2: gaccccctgtgcctct; 2R1: ctccagccaggtccatct; 2R2: tctccagccaggtccat) and exon3 (3L1: tcagcaggctgttgt; 3L2: tcagcaggctgttgtgt; 3R1: ccctggtctcttcat; 3R2: aagcctccctggtctt; 3R3: aagcctccctggtct). The TALENs were constructed with FastTALE TALEN Assembly Kit (Sidansai), and their activities were confirmed in 293T cells as previous description . The constructed TALENs were transfected into 293T cells and selected with 2 μg/ml puromycin (Sigma). The genomic DNA of 293T cells was harvested after selection. Then, PCR and sequencing were performed to examine the efficiency of the TALENs.
Generation of CIITA-deficient hESCs
To prepare the cells for transfection, harvested hESCs were plated in six well plates coated with Matrigel in mTeSR™1 medium (Stemcell Technologies). On the following day the most efficient TALENs (2L2 and 2R2) plasmids and EGFP-Puro plasmid (Sidansai) (1:1:1) were transfected into hESCs by the FuGENE HD transfection reagent (Promega). We incubated the FuGENE HD Transfection Reagent/plasmids/Opti-MEM (Life Technologies) mixture (15 ul/6 ug/300 ul) for 15 min at room temperature, and then the mixture was added into the cell culture. Puromycin was added into media two days later. After selection with 0.5 μg/ml puromycin the survival colonies were dissociated into single cells using TrypLE (Invitrogen) and seeded onto CF1-coated plates at a density of 500 cells/cm2. Two weeks after passaging, the colonies derived from the single cells were transferred into freshly CF1-coated wells, and in parallel, a direct cell PCR kit was used to identify the mutants.
Teratomas formation and derivation of human fibroblasts from teratomas
hESCs were injected intramuscularly into 6–8 weeks NOD/SCID mice (approximately 5 × 106 cells per site). After about 2 months, the tumors were processed for hematoxylin-eosin (HE) staining.
The fibroblast-like cells were also derived from teratomas . Teratomas were cut into pieces with scissors and cultured in DMEM supplemented with 10 % serum, 1 % Pen-Strep, and 50 uM β-mecaptoethanol. After several passages, the adherent cells become homogenous and fibroblast-like cells. Cell morphological observation and RT-PCR were performed (Additional file 2: Figure S1a, b). Ten cell lines were established (3 for +/+; 3 for +/–; 4 for −/−). And we analyzed some mesenchymal stem cells markers in established cells lines (n > 3) (Additional file 2: Figure S1c). CCD and mesenchymal stem cells (MSC) were used as control. Those cell lines were more like fibroblasts. It showed that this method was reproducible in our experiments. All of the animal experiments were conducted in accordance with the Guide for the Care and Use of Animals for Research Purposes and approved by the Zhejiang University Animal Care Committee.
Reverse Transcription-PCR and Real-Time PCR analysis
Total RNA was prepared using an RNeasy kit (Qiagen), which was then used as a template for real-time PCR (RT-PCR). RT-PCR was performed in an Eppendorf Mastercycler® ep realplex real-time PCR system using SYBR Green-based PCR Master mix (TOYOBO). Standard curves were acquired for both the genes of interest and internal control (G3PD). The primers used are listed in Additional file 3: Table 1.
Immunochemistry was performed as previously described . The primary antibodies used were anti-Oct4 (1:100, Santa Cruz Biotechnology), anti-Nanog (1:150, Santa Cruz Biotechnology), anti-Tra-1-60 (1:150, Chemicon), anti-SSEA3 (Ascites, 1:400, Developmental Studies Hybridoma Bank), anti-CIITA (1:200, Santa Cruz Biotechnology) and anti-HLA DR+DP+DQ (1:200, Abcam) for staining.
Protein extracts were obtained by lysing cells. Samples were fractionated by SDS-PAGE and transferred to polyvinylidene-fluoride (PVDC) membrane. After blocking with 5 % milk in PBST (phosphate-buffered saline with 0.1 % tween 20) for 1 h at room temperature, the membranes were probed with the corresponding primary and secondary antibodies. And the following antibodies were used: anti-HLA DR+DP+DQ (1:200, Abcam), anti-CIITA (1:200, Santa Cruz Biotechnology) and anti-β-Actin (1:5000, HuanAn).
Derivation of human DCs from hESCs
We induced the differentiation of DCs from hESCs by step-wise growth factors induction in suspension culture of EBs as previously reported . In the first 5 days, hESCs showed mesoderm specification in the X-VIVO™ 15 medium (Lonza) supplemented with 1 mM sodium pyruvate, 1× non-essential amino acids, 2 mM l-glutamine, 50 mM 2-mercaptoethanol and the four growth factors, including recombinant human bone morphogenetic protein-4 (rhBMP-4; BD), recombinant human vascular endothelial growth factor (rhVEGF; R&D), recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF; R&D) and recombinant human stem cell factor (rhSCF; R&D). From day 6 to day 10, rhBMP-4 was removed, and the cells became hematopoietic stem cells (HSCs). From day 11 to day15, rhVEGF was removed and HSCs turned into common myeloid progenitors (CMP). From day 16 to day 20, rhSCF was removed and monocyte-like cells appeared and accumulated gradually as DC precursors. DC precursors will become immature DCs (iDCs) with the treatment of rhGM-CSF and recombinant human Interleukin 4 (rhIL-4; R&D) in the next 4–6 days. The maturation of DCs needed further incubation for 1–2 days with the mixed factors, including rhGM-CSF, recombinant human Interleukin-1 beta (rhIL1-β; R&D), recombinant human Interferon gamma (rhIFN-γ; R&D), Prostaglandin E2 (PGE-2; Sigma) and recombinant human Tumor necrosis factor alpha (rhTNF-α; R&D).
Fluorescence-activated cell sorting
Cells were dissociated with TrypLE and were stained for 30 min at 4 °C in 100 μL of 0.5 % FBS in PBS containing an appropriate dilution of PE, FITC, APC or PE-CY™7-conjugated antibody. Primary antibodies included human CD83, CD86, HLA II and HLA I (BD Biosciences). The sample measurement was performed on a BD FACSCalibur flow cytometer system, and the analysis was performed using FlowJo software (Tree Star, Ashland, OR).
All of the data are represented as the mean ± SEM. The data were analyzed statistically using GraphPad Prism 5.1 (GraphPad Software Inc., USA). All performance variants were analyzed by the unpaired Student’s T test. Statements of significance were based on *P < 0.05 unless otherwise stated.