Chir99021

iPSC derived endothelial cell differentiation was performed essentially as described [23 (link)]. To induce endothelial cell differentiation, approximately 1 x 10⁵ human iPSCs were seeded in each well of Matrigel-coated 6-well plates and cultured in differentiation medium containing RPMI and B-27 supplement (minus insulin) with 5 μM CHIR-99021 (a glycogen synthase kinase [GSK]-3 inhibitor; Selleck Chemicals, Houston, TX) for 2 days, followed by RPMI/B27 (minus insulin) with 2 μM CHIR-99021 for 2 additional days. The medium was then changed to RPMI/B-27 (minus insulin) with 25 ng/mL vascular endothelial growth factor (VEGF; R&D Systems) and 10 ng/mL bFGF (R&D Systems) for 3 days. Finally, for the subsequent 7 days the medium was changed to RPMI/B27(with insulin) with 25 ng/mL VEGF and 10 ng/mL bFGF. At day 14, human iPSC-ECs were sorted for CD31⁺/CD144⁺ cells using FACS. Antibodies against CD31 and CD144 were obtained from Biolegend.

To initiate the differentiation of mouse iPSCs to ECs, medium was changed to RPMI-1640 containing 2% B-27 minus insulin (both Gibco) and 5 μM CHIR-99021 (Selleckchem). Starting on day 2, RPMI-1640 containing 2% B-27 minus insulin (both Gibco) and 2 μM CHIR-99021 (Selleckchem) was used. From day 4 to day 7, cells were exposed to RPMI-1640 EC medium containing 2% B-27 minus insulin plus 50 ng ml⁻¹ of mouse vascular endothelial growth factor (mVEGF, R&D Systems), 10 ng ml⁻¹ of mouse fibroblast growth factor basic (mFGFb, R&D Systems), 10 μM Y-27632 (Sigma-Aldrich) and 1 μM SB 431542 (Sigma-Aldrich). EC clusters were visible from day 7, and cells were maintained in Endothelial Cell Basal Medium 2 (PromoCell) plus supplements, 10% FCS heat-inactivated (HI) (Gibco), 1% penicillin–streptomycin, 25 ng ml⁻¹ of VEGF, 2 ng ml⁻¹ of FGFb, 10 μM Y-27632 (Sigma-Aldrich) and 1 μM SB 431542 (Sigma-Aldrich). The differentiation process was completed after 21 days, and undifferentiated cells were detached during the differentiation process. For purification, cells went through MACS purification using anti-CD15 mAb-coated magnetic microbeads (Miltenyi) for negative selection.

Human ESC lines H1 (Wi Cell), H9 (Wi Cell) and knockout cell lines were maintained in mTeSR1 (STEMCELL Technologies) on matrigel (Corning)-coated plates. Mouse ESC cell line OG2 with GFP controlled by Oct4 promoter, was kindly provided by Dr. Jiekai Chen. OG2 mESCs and knockout mESCs based on OG2 mESCs were maintained on feeder layers in mESC + 2iL medium (DMEM/ high glucose (Hyclone), 15% FBS (Gibco), NEAA (Gibco, 100×), GlutaMAX (Gibco, 100×), Sodium Pyruvate (Gibco, 100×), 1 μM PD0325901 (Selleck), 3 μM CHIR99021 (Selleck), 1000 units/mL mLIF). OG2 mESCs and knockout mESCs based on OG2 mESCs were maintained on gelatin (Millipore)-coated plate in mouse N2B27 + 2iL medium (50% DMEM/High glucose (Hyclone), 50% Knockout DMEM (Gibco), N2 (Gibco, 200×) + B27 (Gibco, 100×), NEAA (Gibco, 100×), GlutaMAX (Gibco, 100×), Sodium Pyruvate (Gibco, 100×), 1 μM PD0325901 (Selleck), 3 μM CHIR99021 (Selleck), 100 μM β-mercaptoethanol (Gibco), 1000 units mL⁻¹ mLIF). All cell types were maintained at 5% CO₂.

Undifferentiated human ESC HUES8 and iPSC PGP1 were maintained in mTeSR1 (StemCell Technologies, Cat# 85850) on Matrigel-coated cell culture plates at 37 °C with 5% CO₂. For directed differentiation of DE, human PSCs were cultured in DMEM-F12 or DMEM medium supplemented with 0.2% bovine serum albumin (Yeasen Biotechnology, Cat# 36101ES76), 100 ng/mL Activin A (PeproTech, Cat# 120-14P), 2.5 μM CHIR99021 (Selleck, Cat# S2924) for 24 h, then the CHIR99021 was removed from a medium for the following 3–4 days. Next, the differentiation of the pancreatic progenitor cells was performed according to a previous report [47 (link)]. Small molecules used in this study are listed as follows: XCT790 (XCT), 2–5 μM (MCE, Cat# HY-10426); ATN-224 (ATN), 5–10 μM (MCE, Cat# HY-16074); Mdivi-1, 10 μM (MCE, Cat# HY-15886); IACS-010759 (IACS), 0.1–20 nM (Selleck, Cat# S8731); Dynasore (DYNA), 1–3 μM (Selleck, Cat# S8047); FCCP, 1–2 μM (Selleck, Cat# S8276); Oligomycin A (Oligo), 0.1–10 μM (Apexbio, Cat# A5588); Doxycycline (DOX), 100 ng/ml (Sigma, Cat# 324385). Of note, the concentrations of the chemicals we used in this study showed little effect on the survival of cells.

The MTT assay was used to measure MenSCs viability. MenSCs (1000/well) were seeded in 96-well plates overnight. To detect the effects of rapamycin on MenSCs viability, cells were incubated with different concentrations (1, 10, 50, and 300 nM and 1 μM) of rapamycin for 24 h and different time with 50 nM or 1 μM rapamycin (Selleck, Cat#S1039, USA) for 2, 4, 6, and 8 days; normal culture media with a proportionate DMSO were used for the control group.
To detect the beneficial effects of CHIR99021 (Selleck, Cat#S1263, USA) on MenSCs treated with rapamycin, cells were first co-cultured with CHIR99021 (1 μM) and different concentrations (1, 10, 50, and 300 nM and 1 μM) of rapamycin for 24 h; normal culture media with a proportionate DMSO were used for the control group.
To detect the proliferation rates of shGFP, shATG5, and shGSK3β MenSCs, cells (1000/well) were seeded in 96-well plates overnight. All the groups are cultured with normal culture media.
At the prespecified time points, 10 μl of MTT solution (Songon, Cat#A600799, China) was added to the cells. After incubation for another 4 h and DMSO for 5 min, the optical density (OD) values were determined at 490 nm using a microplate reader. Each group was tested in triplicate for five replicate wells.

Lung cancer cells (NCI-H460) were cultured with HUVECs seeded at a density of 1 × 10⁶ cells/well in an ultra-low attachment 6-well plate (Corning B.V. Life Sciences, Amsterdam, Netherlands). After 2 days, MCTSs were implanted subcutaneously in male BALB/c-nu mice and allowed to grow to a tumor volume of 100–200 mm. Tumor-bearing mice were randomized into the following four groups of six mice each; Group 1, control with normal saline (N/S); Group 2, treated with CHIR-99021 (Selleckchem, Houston, TX, USA), 16 mg/kg, intraperitoneal injection (i.p.); Group 3, treated with Gefitinib (Selleckchem, Houston, TX, USA), 50 mg/kg, oral injection (p.o.); Group 4, treated with CHIR-99021 and Gefitinib, respectively. All drugs were administered 3 days a week for 2 weeks. All treated mice were sacrificed on day 28, and tumors were resected for further histological evaluation.