The hPSC cell line, 83iCTR-n1, and the human embryonic stem cell line, H9 were obtained from the induced pluripotent stem cell (iPSC) core laboratory, Cedars-Sinai Regenerative Medicine Institute. These cells were negative for mycoplasma in the same facility before being used for differentiation experiments. At the core laboratory, hPSC derivation from human fibroblasts was performed according to published protocols [
31,
32]. The hPSC line was used at passage 40–50; H9 cells were used at passage 20–30. Both cell lines were cultured in a feeder-free system with mTeSR1 basal medium plus 5× supplement (Invitrogen, Carlsbad, CA, USA), 200 μmol/l
l-alanyl-
l-glutamine (American Type Culture Collection [ATCC], Manassas, VA, USA) and 0.1 mmol/l β-mercaptoethanol (STEMCELL Technologies, Vancouver, BC, Canada). EBs were generated according to the manufacturer’s instructions and maintained in AggreWell medium (STEMCELL Technologies) supplemented with 10 μmol/l rho-associated protein kinase (ROCK) inhibitor (Sigma-Aldrich, St Louis, MO, USA). The human microvascular EC (HMEC) line was purchased from E. Ades and F. J. Candal (Centers for Disease Control and Prevention, Atlanta, GA, USA), and T. Lawley (Emory University, Atlanta, GA, USA). HMECs were negative for mycoplasma and were grown at 37°C under 5% CO
2 and maintained in MCDB131 medium (Invitrogen) supplemented with 1%
l-alanyl-glutamine (vol./vol.) (ATCC), 10% FBS (vol./vol.) (Omega Scientific, Tarzana, CA, USA) and 100 μg/ml endothelial cell growth supplement (Millipore, Hayward, CA, USA), for use at passages 20–25. For co-culture experiments, 100 EBs were picked up with a glass Pasteur pipette and 5 × 10
5 HMECs inactivated with 100 μl/ml mitomicyn C (Sigma-Aldrich) were added to collagen I solution (BD Bioscience, Franklin Lakes, NJ, USA) with 1× minimum essential media (MEM), 1 mol/l 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer, 7.5% bicarbonate solution (vol./vol.) (Life Technologies, Grand Island, NY, USA), 0.1 mmol/l NaOH, 1 mg/ml laminin I and 1 mg/ml collagen IV (R&D Systems, Minneapolis, MN, USA) and placed on ice. Then, 10 × 100 μl drops of cell suspension were placed into Petri dishes (60 × 20 mm; VWR, Cerritos, CA, USA) and solidified at 37°C for 10–20 min, after which, medium containing pancreatic differentiation factors including activin A, Wnt3a, retinoic acid (RA), fibroblast growth factor 7 (FGF7), epidermal growth factor (EGF), SB461542, hepatocyte growth factor (HGF), insulin-like growth factor-1 (IGF-1), and nicotinamide was added at different time points of EB development [
7]. EB controls were cultured in gel without ECs but treated with the same pancreatic differentiation factors. After 20 days, cells were harvested using 5% collagenase I (wt/vol.) (Worthington, Lakewood, NJ, USA) and maintained in CMRL 1066 medium with CIT modification (Mediatech, Manassas, VA, USA), supplemented with 10 ml of 25% human serum albumin (HSA; NOVA Biologics, Oceanside, CA, USA) to a final concentration of 0.5% (vol./vol.), and 50 μl of a 1 mg/ml stock of IGF-1 (R&D Systems) at a final concentration of 10 ng/ml (vol./vol.). To generate definitive endoderm (DE) cells, undifferentiated iPSCs were cultured in a feeder-free system and culture medium, as described above. Subsequently, cells were transferred into six wells of a 24-well plate. At 90% confluency, RPMI 1640 (Life Technologies) containing 100 ng/ml activin A (Peprotech, Rocky Hill, NJ, USA) and 25 ng/ml Wnt3a (R&D Systems) was added to DE cells for the first day. On the second and third days, the cell medium was changed to RPMI 1640 containing only 100 ng/ml activin A. The DE cells were plated in collagen gel (as described above) with or without ECs, and pancreas differentiation factors were added [
7]. The human hepatoma cell line and beta-TC-6 cells (ATCC) were both negative for mycoplasma.