Dulbecco s modified eagle medium nutrient mixture f 12

HEC1B and KLE type II human endometrial cancer cell lines were purchased from the American Type Culture Collection (Manassas, VA) [61 (link)]. HEC-1B cells were cultured in Minimal Essential Medium (Gibco, Life Technologies, Burlington, ON) supplemented with 10% fetal bovine serum (FBS; Hyclone Laboratories Inc., Logan, UT). KLE cells were cultured in Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (Gibco, Life Technologies) supplemented with 10% fetal bovine serum (FBS; Hyclone Laboratories Inc.). Cultures were maintained at 37°C in a humidified atmosphere of 5% CO2 in air.

Mouse bone marrow‐derived mesenchymal stem cells (mMSC) were obtained from Life Technologies (Grand Island, NY, Lifetechnologies.com) and grown in Dulbecco's modified Eagle medium/Nutrient Mixture F‐12 containing MSC‐Qualified fetal bovine serum (Gibco, Grand Island, NY, thermofisher.com). Human bone marrow‐derived mesenchymal stem cells (hMSC) were obtained as previously described 21, 22, 23, 24. Nonmalignant human hepatocytes (HH) were obtained from Sciencell (Carlsbad, CA, sciencell.com) and cultured as recommended by the supplier.

NP cells were isolated in accordance with a previous study [31 (link)]. Briefly, NP tissues were cut into pieces and enzymatically digested in 0.2% type II collagenase for 3 h. After washing and centrifugation, the isolated cells were cultured in Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (Gibco) containing 15% fetal bovine serum (Gibco). NP cells from the second passage were used for the subsequent experiments. Previous studies have shown that the elastic modulus of healthy NP tissues is 0.3–5 kPa, that of mildly degraded NP tissues is 10–17 kPa, and that of severely degraded NP tissues is 20–25 kPa [19 (link), 32 (link), 33 (link)]. To build the gradient stiffness model, NP cells were cultured for 48 h in 1 kPa (soft), 12 kPa (moderate) and 25 kPa (stiff) polystyrene plates (Matrigen, Brea, CA). The Matrigen plates are commercially available produces with variant stiffness and biocompatibility, which enables researchers to study matrix stiffness on cell behaviors [34 (link), 35 (link)].

ES cells were induced to differentiate into neuronal lineages as previously described, with some modifications^{23 ,34 (link)}. For single-cell suspensions, the cells were dissociated from monolayer culture (day 0) with 0.25% trypsin–EDTA (trypsin–EDTA (0.5%), Gibco, Cat.No 15400–054). The 8 days old EBs were dissociated and plated and after two days the medium was changed to 1:1 DMEM/F12 (Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12, Gibco, Cat.No 31331-028): Neurobasal medium (Neuronal Base Medium For Neuronal Cells, Gibco, Cat.No 21103-049) with FBS (Foetal Bovine Serum, APS, Cat.No S-001A, USDA grade), 1 mM GlutaMax, 3 mg/ml AlbuMax I, 50U/ml penicillin/streptomycin, 0.5% (vol/vol) N-2 Supplement (N-2 Supplement (100x), Gibco, Cat.No 17502-048), and 1% (vol/vol) B-27 Supplement (B-27 Supplement (50x), Gibco, Cat.No 17504-044). Neuroectoderm formation was induced by all-trans retinoic acid (atRA) treatment between day 4 and 8 of differentiation. At day 8 the EBs were collected, trypsinized to single cell suspension, then seeded onto surface coated tissue culture dishes and differentiated further for 6 more days (Fig S2).