Rhnoggin

For neural induction, hiPSC colonies were detached with Accutase (Life Technologies), suspended as single cells, and plated on 0.1% gelatin-coated wells (Sigma-Aldrich) for 1 h to allow MEFs to attach. Floating iPSCs were then collected and plated on Matrigel (BD Pharmingen)-coated dishes (50,000 cells/cm²) in MEF preconditioned iPSCM in the presence of 10 μM of the Rho kinase inhibitor Y-27632. When the cell culture reached ≈90% confluence (usually 2 d after plating), the culture medium was replaced with KO serum replacement medium supplemented with 200 ng/ml of rhNOGGIN (R&D) and 10 μM of SB431542 (Sigma-Aldrich). The medium was changed daily for the next 3 d. Thereafter, it was switched every other day to gradually expose the cells to increasing (1:3, 1:1, 3:1) ratios of NSC/KO serum replacement medium. 2 d after the final switch, the cells were detached using Accutase and plated on Matrigel-coated dishes in neural stem cell medium (NSCM) supplemented with 20 ng/ml basic fibroblast growth factor and 20 ng/ml EGF in the presence of 10 μM of the Rho kinase inhibitor Y-27632, according to the published Dual Smad inhibition protocol (Chambers et al., 2009 (link)).

CRPC with docetaxel chemotherapy-resistant patient-derived organoids was established pursuant to the method as described previously [51 (link),52 (link)]. The organoids were cultured with the advanced DMEM/F12 supplemented with 125 ng/ml rhR-spondin-1 (STEMCELL Technologies, 78213.1), 100 ng/ml rhNoggin (R&D Systems, 6057-NG-025), 1 ng/ml rhFGF (R&D Systems, 233-FB-025), rhFGF-10 (R&D Systems, 345-FG-025), 50 ng/ml (Meilunbio, China, MB8218-1), 1×N21 (R&D Systems, AR008), 10 μM Y-27632 (Abcam, Ab120129), 0.5 μM A83-01 (Beyotime Biotechnology, SF7917), 1:100 primocin (Invivogen, ant-pm-1), 10 μM SB202190 (Beyotime Biotechnology, SC0380), 10 mM Nicotinamide (Beyotime Biotechnology, S1761), and 1.25 mM N-acetylcysteine (Selleck, S1623), and the medium was changed every 2 to 3 days. Lentiviral transduction of AZGP1P2 shRNA and shRNA control was conducted according to the method as previously described [53 (link)]. The proliferation of organoids treated with 20 nM docetaxel was assessed by CCK-8 assay on the fifth day, and organoids were photographed using light microscopy. Three independent experiments were performed.

CRPC with docetaxel chemotherapy-resistant patient-derived organoids was established pursuant to the method as described previously [51 (link),52 (link)]. The organoids were cultured with the advanced DMEM/F12 supplemented with 125 ng/ml rhR-spondin-1 (STEMCELL Technologies, 78213.1), 100 ng/ml rhNoggin (R&D Systems, 6057-NG-025), 1 ng/ml rhFGF (R&D Systems, 233-FB-025), rhFGF-10 (R&D Systems, 345-FG-025), 50 ng/ml (Meilunbio, China, MB8218-1), 1×N21 (R&D Systems, AR008), 10 μM Y-27632 (Abcam, Ab120129), 0.5 μM A83-01 (Beyotime Biotechnology, SF7917), 1:100 primocin (Invivogen, ant-pm-1), 10 μM SB202190 (Beyotime Biotechnology, SC0380), 10 mM Nicotinamide (Beyotime Biotechnology, S1761), and 1.25 mM N-acetylcysteine (Selleck, S1623), and the medium was changed every 2 to 3 days. Lentiviral transduction of AZGP1P2 shRNA and shRNA control was conducted according to the method as previously described [53 (link)]. The proliferation of organoids treated with 20 nM docetaxel was assessed by CCK-8 assay on the fifth day, and organoids were photographed using light microscopy. Three independent experiments were performed.

hESC H1 line was cultured feeder-free on hESC-qualified Matrigel (BD Biosciences) in E8 media (Stemcell Technologies) with 30% of irradiated mouse embryonic fibroblasts (iMEFs) conditional media. Cells were passaged every 3–5 days at 80% confluent with TrypLE Express (Invitrogen). After dissociation, the cells were plated in E8 media with 10 μM Y-27632, (StemGent) for 24 h. After 24 h media without Y-27632 was replenished daily. iMEF conditional media was prepared by incubating iMEFs with hESC media without bFGF for 24 h for 7 days. Collected media were filtered, flash frozen and stored at −80 °C. To initiate differentiation, the cells were dissociated using TrypLE Express to single cells and seeded at 150,000 cell/cm2 onto 1:30 dilution of growth factor reduced Matrigel (BD Biosciences) in DMEM/F12 in E8-MEF conditional media with 10 µM Y-27632. Two days following seeding the differentiation was started. Day 1 cells were exposed to RPMI + 3 µM CHIR-99021 (Stemgent) + 100 ng/ml rhActivinA (R&D Systems). Days 2–3: + 100 ng/ml rhActivinA + 0.2% FBS. Day 4–5: + 2% FBS + 50 ng/ml KGF (Peprotech). Days 6–9: DMEM/B27 + 50 ng/ml KGF + 2 μM RA (Sigma) + 0.25 μM SANT-1 (Sigma) + 100 ng/ml rhNoggin (R&D Systems). Days 10–14: DMEM/B27 + PdBU (1 µM) + Alk5i (1 µM) + 100 ng/ml rhNoggin (R&D Systems). PPs are defined as Day 9 and EPs as Day 14 of differentiation, respectively.