D0627

Immortalized HESC line was purchased from the American Type Culture Collection (ATCC CRL-4003) and cultured according to the manufacturer’s instructions (Krikun et al., 2004 (link)). The identity of the cell lines has been authenticated by ATCC, and we have confirmed that the cell lines tested negative for mycoplasma contamination. Briefly, HESC was cultured in Dulbecco’s modified Eagle’s medium (DMEM)/F12 (Gibco) supplemented with 1% penicillin and 1% streptomycin, 10% charcoal-stripped fetal bovine serum (CS-FBS, Biological Industries), 3.1 g/l glucose, 1 mM sodium pyruvate, 1% Insulin-Transferrin-Selenium (ITS) (Gibco), and 500 ng/ml puromycin at 37°C in a humidified chamber with 5% CO₂. To induce decidualization in vitro, HESC was treated with differential medium (DMEM/F12 with 2% CS-FBS) containing 1 μM medroxyprogesterone 17-acetate (MPA, Sigma, M1629), and 0.5 mM dibutyryl cAMP (dbcAMP, Sigma, D0627). The medium was changed every 48 hr. Other reagents including inhibitors, activators are listed in Supplementary file 1B.

A safe and effective in vitro ovarian culture system has been established in our lab (10 (link), 11 (link)). Neonatal mice were sacrificed by cervical dislocation at the designated times. The ovaries were separated in cold phosphate-buffered saline (PBS) under the microscope. The isolated ovaries were incubated in 6-well culture dishes (NEST, China), and an insert (PICM0RG50, Millipore, USA) was placed in every well with 3 mL Dulbecco Modified Eagle Medium/Ham F12 nutrient mixture (DMEM/F12) (Gibco, Life Technologies, CA) supplemented with insulin-transferrin-sodium selenite (1:100, Sigma, USA) and penicillin-streptomycin solution at 37 °C, 5% CO₂ and saturated humidity. Ovaries were randomly assigned, and cultured for 1–7 days in basal medium alone or basal medium supplemented with either dbcAMP (10 μM, D0627, Sigma, USA), MDL-12,330 (5 μM, M182, Sigma, USA), Milrinone (10 μM, 78415-72-2, J&K, China) or H 89 2HCL (5 μM, S1582, Selleck, China), respectively.

Cells were inoculated into the indicated medium at a density of 5 × 10⁵ cells per ml from a YPD overnight culture. Medium amended with 10 mM dbcAMP from a 100 mM stock solution of dbcAMP (D0627; Sigma) in water. Cultures were incubated at 37°C for 3 h in glass-bottom 12-well dishes and then fixed with 0.37% formaldehyde, and morphological assessments by DIC microscopy were performed on an inverted microscope.

The murine N9 microglial cell line was provided by Dr Paola Ricciardi-Castagnoli (Singapore Immunology Network, Agency for Science, Technology and Research, Singapore). The N9 microglial cells were cultured in Roswell Park Memorial Institute medium (RPMI 1640; Invitrogen, 21,875-091) supplemented with 10% FBS, 2mML-Glutamine (Sigma, G6392),100 U/ml penicillin, and 100 mg/ml streptomycin, and the cultures were maintained at 37°C, 95% air, and 5% CO₂ in a humidified incubator [67 (link)]. The cells were then seeded onto 35-mm culture dishes (0.5 x10⁶ cells/ well) for analysis.
Dopaminergic MES 23.5 cells, a gift from Dr Wei-dong Le (Baylor College of Medicine, TX, USA), were cultured in DMEM/F12 containing Sato components growth medium supplemented with 2% fetal bovine serum (FBS), 100 units/ml penicillin, and 100 mg/ml streptomycin at 37°C in a humidified CO₂ incubator (5% CO₂, 95% air) [68 (link)]. For experiments, MES 23.5 cells were plated at a density of 0.5 × 10⁵/cm² onto 35-mm plastic dishes, glass coverslips, previously coated with poly-L-ornithine (P-4638, Sigma; 10 mg/ml). Cells were stimulated to enhance differentiation by adding dibutyryl-cAMP (D0627, Sigma; 1 mM) to the supplemented growth medium, and they were grown to 80% confluence before the start of any treatment.

0.5 μg/ml doxycycline (D9891, Sigma-Aldrich) was used to induce Neurog1 and -2 genes. After 4 dpi, half of the mTeSR 1 media was replaced with supplemented BrainPhys media (05790, StemCell Technologies), replaced daily until 7 dpi and further once per week. The following BrainPhys supplements were used for 10 ml media: 200 μl NeuroCult™ SM1 Neuronal Supplement (05711, Stemcell Technologies); 100 μl N2 Supplement-A (17502048, Thermo Fisher Scientific); 20 μl of 10 μg/ml recombinant human BDNF to a final concentration of 20 ng/ml (450-02, Peprotech); 20 μl of 10 μg/ml recombinant human GDNF to a final concentration of 20 ng/ml (450-10, Peprotech); 98 μl of 50 mg/ml dibutryl-cAMP to a final concentration of 1 mM (D0627, Sigma-Aldrich); 50 μl of 40 mM ascorbic acid to a final concentration of 200 nM (A0278, Sigma-Aldrich); and 100 μl of 100× penicillin-streptomycin (15140122, Thermo Fisher Scientific). After stable integration of the Neurog1/2 cassette to CRTD5 cells, the cells were differentiated similarly to iNGN cells.