Esgro

The murine embryonic stem cell lines used in this study include: RW4 (wildtype), CFG37 (wildtype, express the βACT-GFP transgene) (Moore et al., 2009 (link); Okabe et al., 1997 (link)), Ncad19 (N-cadherin (+/−)), Ncad95 (N-cadherin (−/−)) (Moore et al., 1999 (link)), and 9J (E-Cadherin (−/−)) (Larue et al., 1996 (link)). The cells were normally maintained on feeder layers of irradiated murine embryonic fibroblasts in ES cell medium supplemented with 1,000 units/ml of recombinant LIF (ESGRO, Chemicon International), at 37°C and 5% CO₂.
The unlabeled cells were marked by incubating with CellVue Claret reagent (Molecular Targeting Technologies, Inc.) according to the manufacturer's instructions. ES cells were differentiated into endoderm by exposure to 1 µM all trans retinoic acid for 4–7 days as monolayers cultured in gelatin-coated tissue culture dishes. Typically, 80 to 90% of the cells are differentiated as indicated by strong GATA4 or Dab2 expression detected by immunofluorescence microscopy.

ES(MC1R(20)) cells were a kind gift from Minoru Ko. The plvx-Tight-Puro-HA/FLAG H3.3 expression construct was generated as described in [13 (link)]. Lentiviral particles were packaged in 293 T cells with the psPAX2 packaging plasmid. Subsequently, we transduced ES(MC1R(20)) cells and drug-selected with puromycin for stable integration. ESCs were cultured as in [45 (link)]. Briefly ESCs were maintained in medium consisting of Dulbecco’s modified Eagle’s medium (Hyclone) supplemented with 10% fetal bovine serum (FBS), 10% knockout serum replacement (Invitrogen), L-glutamine, 100 units/mL penicillin, 100 μg/mL streptomycin, 0.1 mM beta-mercaptoethanol at 37°C under 5% CO2, supplemented with 1,000 units/mL leukemia inhibitory factor (LIF) (ESGRO, Chemicon). For differentiation into embryoid bodies (EBs), ESCs were trypsinized and transferred to ultra-low attachment plates into 15% FBS containing medium without the addition of LIF and medium was changed every second day.

All the animal experiments were performed following the ethical guidelines approved by Tianjin Animal Management Committee. MEF cells were derived from E13.5 embryos isolated from B6C3F1 mice via cesarean section and washed in phosphate-buffered saline (PBS). Heads and visceral tissues were removed, and the remaining tissue was washed in PBS, submerged in 0.25% trypsin–EDTA (0.25% TE, Invitrogen), and incubated at 37 °C for 10 min. The tissue was pipetted repeatedly to aid dissociation, washed, and plated in MEF medium, Dulbecco’s modified eagle’s medium (DMEM, Invitrogen) supplemented with 10% fetal bovine serum (FBS, Hyclone), 1 mM L-glutamine (Invitrogen), 1% nonessential amino acid stock (NEAA, Sigma), penicillin (100 U/mL), and streptomycin (100 μg/mL). Cells were cultured at 37 °C in 5% CO₂ with humidified air (Thermo Scientific, USA). ESC lines were established and characterized based on a previously described method [44 (link)], cultured in KnockOut DMEM supplemented with 20% FBS (ES quality, Hyclone), 1000 U/mL leukemia inhibitory factor (LIF) (ESGRO, Chemicon), 0.1 mM nonessential amino acids, 0.1 mM β-mercaptoethanol, 1 mM L-glutamine, penicillin (100 U/mL), and streptomycin (100 μg/mL).

iPSCs were generated using the same protocol reported previously [14 (link)]. MEFs obtained from OG2^+/-/ROSA26^+/- double-transgenic mice which were carrying Oct4-GFP and neo/lacZ were infected with retroviruses encoding 4 transcription factors (Oct4, Sox2, Klf4, and c-Myc). After retroviral infection, Oct4-GFP-positive colonies were transferred onto inactivated MEF feeder layers, trypsinized, re-plated, and cultured in ESC medium (Dulbecco's modified Eagle's medium (DMEM; Gibco) supplemented with 15% fetal bovine serum (FBS; Gibco), 1× penicillin/streptomycin/glutamine, 1 mM nonessential amino acids (NEAA; Gibco), 0.1 mM β-mercaptoethanol (Gibco), and 1000 U/ml leukemia inhibitory factor (LIF; ESGRO, Chemicon) [14 (link)].

E14 and 46C ES cells (ES cell line in which EGFP is substituted into the ORF of the Sox1 gene, a reporter for differentiation into neural precursor cells) were cultured in mouse ES (mES) cell medium (DMEM (Gibco) supplemented with 15% FBS, 2 mM GlutaMAX (Gibco), MEM nonessential amino acids, β-mercaptoethanol (Gibco), tylosin, and 1% Pen/Strep (Gibco)) supplemented with LIF (ESGRO, Chemicon) on 0.2% gelatin-coated dishes. Cells were maintained at 37°C in a humidified atmosphere of 5% CO₂.

Mouse iPSC were generated and maintained in Dulbecco's Modified Eagle's medium (DMEM) with 1000 IU/ml leukemia inhibitory factor (LIF, Chemicon, ESGRO), as described previously [11] . Mouse embryonic fibroblasts (MEF) obtained from embryos at 14 days post-coitum were prepared and treated with mitomycin-C (10 µg/mL) to control MEF overpopulation. Embryoid body (EB) formation was promoted by placing iPSC in 25 µl hanging drops (∼250 cells per drop) and culturing the suspension in iPSC medium without LIF. After 5 days, EBs were transferred to 0.1% gelatin-coated dishes. Medium was changed the following day and then changed every other day to maintain viable cells. Cells were then allowed to differentiate into large aggregates, using differentiation media and growth factor conditions reported previously by Stevens et al. [12] (link) to induce differentiation into CM. Cell culture conditions were further modified to optimize directed differentiation into precursor CM with lentiviral genomic manipulation.