Human activin a

DF-1 cells were received from the cell bank of the Friedrich-Loeffler-Institut and cultured in DMEM medium supplemented with 2 mM L-glutamine, 0.1 mM mercaptoethanol, 0.4 mM sodium pyruvate, non-essential amino acids, 10% fetal calf serum, and 1× penicillin/streptomycin (D10). Chicken primary fibroblasts were derived from day 10 (E10) embryos and maintained in D10 medium.
PGCs were derived from chicken embryonic gonads (E7, stage HH 28–29) and cultured as described^{49 (link)}. The PGCs were cultured in suspension without a feeder-layer in customized avian KO-DMEM (23). Shortly, a CaCl₂-free medium (12.0 mM glucose, 250 mOsm) produced by ThermoFisher-scientific was used as the basal medium. It was supplemented with 1× B-27, 2.0 mM GlutaMax, 1× NEAA, 0.1 mM ß-mercaptoethanol, 1× nucleosides, 0.4 mM pyruvate, 0.2% ovalbumin (Sigma), 0.1 mg/ml sodium heparin (Sigma), 0.15 mM calcium chloride, 12.5 ng/ml human activin A (PeproTech), 4 ng/ml basic fibroblast growth factor (PeproTech), and 0.2% chicken serum (ThermoFisher scientific). All cells were cultured at 37 °C and 5% CO₂. For the transfections, total 1.5 × 10⁶ cells were suspended in 200 µl R-buffer with 10 µg plasmid-DNA. For transfections, the Neon Transfection System (ThermoFisher scientific) was used (1700 V, 20 ms, 1 pulse).

Avian PGC culture medium contained B-27 supplement, 2.0 mM GlutaMax, NEAA, 0.1 mM β-mercaptoethanol, nucleosides, 1.2 mM pyruvate, 0.2% ovalbumin (Sigma), 0.2% sodium heparin in avian DMEM, and a custom basal medium (a modification of knockout DMEM (250 mosmol/L, 12.0 mM glucose, and CaCl-free)) [17 (link)]. The following growth factors were added before use: human Activin A, 25 ng/mL (Peprotech); human FGF2, 4 ng/mL; 0.2% ovotransferrin.

EpiSCs were routinely cultured in N2B27/Activin/bFGF: DMEM/F-12 (Gibco, 21331-020) medium supplemented with N2, B27, human Activin A (20 ng/ml; Peprotech) and bFGF (12 ng/ml; Invitrogen). For N2B27/2i/LIF, DMEM/F-12 (Gibco, 21331-020) medium were supplemented with N2, B27, LIF (1000 U/ml), the ERK inhibitor PD0325901 (1 μM; Stemgent) and the GSK3b inhibitor CHIRON99021 (3 μM; Stemgent). In the RT-qPCR Nanog expression analysis, Nanog mRNA levels were quantitated by qRT-PCR 48 h after transfection of TALE-As or dCas9-As/gRNAs and Dox induction. For EpiSC reprogramming, the culture medium was changed from N2B27/FGF/Activin to N2B27/2i/LIF (or 2i/LIF) for 14 days, 2 days after TALE-A and dCas9-A expression so as to maintain and select for induced pluripotent stem cells.