Matrigel hc

hiPSCs were routinely cultured in 6-well plates on 1:75 diluted Matrigel™ HC (Corning #354263), in FTDA medium [10 (link)]. FTDA consisted of DMEM/F12, 1× PenStrep/l-glutamine, 1× defined lipids (Life Technologies #21331020, #10378016, and #11905031, respectively), 0.1 % human serum albumin (Biological Industries #05-720-1B), 1× ITS (BD #354350), 10 ng/ml FGF2 (PeproTech #100-18B), 0.2 ng/ml TGFβ1 (eBioscience #34-8348-82), 50 nM Dorsomorphin (Santa Cruz #sc-200689), and 5 ng/ml Activin A (eBioscience #34-8993-85). Cells were routinely passaged as single cells or, initially, as clumps of cells. For single cell splitting, cells were grown to full confluence (until cultures seemingly appeared syncytial), digested for 10–15 min using Accutase™ (Millipore #SCR005) with 10 µM Y27632 (abcamBiochemicals #ab120129), and replated in the presence of 10 µM Y27632 at 400,000–600,000 cells per well of a 6-well plate. hiPSCs reached confluence after 3 days under these conditions and were subsequently harvested as above, for continuous maintenance or for the induction of differentiation. hiPSCs were kept in culture for a maximum of 30 passages. Cell lines were tested negative for mycoplasma.

Male NMRI-Foxn1^nu/Foxn1^nu and female SCID CB-17/Icr-Prkdc^scid/Rj mice were transplanted with human insulin-producing EndoC-βH1 cells as described^{44 (link),47 (link)}. Male NMRI-Foxn1^nu/Foxn1^nu mice were used for methodological development/validation, but all follow up experiments were performed in female SCID CB-17 mice. At the day of inoculation, 4–6 × 10⁶ EndoC-βH1 cells were seeded on a rubber toric joint (EFJM), supported in Matrigel HC (Corning) supplemented with MmVEGF-164 (1 ng/ml) (BioLegend). The cell-containing or the empty vehicle rubber rings were then inserted under the epimysium in the biceps or quadriceps femoris muscle. The mice were anesthetized with 3 % isoflurane. They received short-term analgesic (Buprenorphine 0.1 mg/kg) and long-term analgesic (3 mg/ml of Acetaminophen-supplemented water for 10 consecutive days) respectively before and after the surgery. Random glycaemia was measured weekly with an ACCU-CHEK Nano glucometer (ROCHE). Once the tumour became palpable, the mice received 20 % glucose-supplemented drinking water to counter the progressive hypoglycemia induced by the EndoC-βH1 cells. Human C-peptide was measured in plasma with a human Ultrasensitive C-peptide ELISA (Mercodia). Kelly tumour-bearing mice were generated by s.c. injections of 5 × 10⁶ cells mixed 1:1 with Matrigel (Corning) in the hind leg of female Crl:NU(NCr)-Foxn1^nu mice.

WT hESCs (HuES6^{29 (link)} background, obtained from Harvard University), WT hiPSCs (F1 background, fetal liver fibroblast-derived^{21 (link)}), and their genetically modified derivatives were maintained on 6-well dishes coated with 1 ml/well 1:75 diluted Matrigel™ HC (Corning #354263), in defined FTDA medium^{30 (link)}. FTDA was composed of DMEM/F12, 1 × PenStrep/Glutamine, 1 × defined lipids (Thermo), 1 × ITS (Corning), 0.1% human serum albumin (Biological Industries), 10 ng/ml FGF2 (PeproTech #100-18B), 0.2 ng/ml TGFβ1 (eBioscience #34-8348-82), 50 nM Dorsomorphin (Santa Cruz), and 5 ng/ml Activin A (eBioscience #34-8993-85). Fully confluent hPSC cultures were harvested by a 15–20 min incubation with Accutase™ (Sigma) containing 10 µM ROCK inhibitor Y-27632 (abcamBiochemicals) and seeded out for passaging into new 6-well plates at 400,000–500,000 cells per well, in FTDA + ROCKi. Cells were split every 3–4 days and kept in culture for a maximum of 30 passages. Short-term signaling stimulation experiments were carried out using semiconfluent cultures.

Four PTC iPSC and four TTF iPSC lines were assessed for their ability to form teratoma. Induced pluripotent stem cells were grown under standard conditions until approximately 70–80% confluent. Cells were dissociated into single cells, and 4–8.5 million cells resuspended in Matrigel HC (Corning) were injected into 6–8 week old male NOD/SCID mice (Charles River). Teratomas were collected, fixed in 10% neutral buffered formalin (NBF), dehydrated and embedded in paraffin blocks. Teratoma analysis and interpretation were performed by a trained pathologist at The Centre for Phenogenomics (Toronto, ON).

LNCaP prostate cancer cells were cultivated as monolayer cultures in RPMI medium (Sigma-Aldrich, St. Louis) supplemented with 10% fetal bovine serum (Sigma-Aldrich) at 37 °C, 95% humidity and 5% CO₂. LNCaP spheroids were generated by seeding of LNCaP cells (5,000–25,000 per well) in ultra-low attachment 96-well plates (Corning, New York). Medium was replaced every 48 h. The cell count and viability of spheroids was determined with Luna II Automated Cell Counter (Logos Biosystems) after dissociating spheroids with a 100 µl pipette. For orthotopic injection of single cell suspensions, the cells were harvested, counted, and suspended in a 1:1 mixture of Matrigel HC (Corning) and RPMI culture medium at a density of 40,000 cells/10 μl and preserved on ice until injection. Immediately prior to intraprostatic injection, the suspension was redissolved and aspirated air-free into a cooled 10 μl Hamilton syringe (Hamilton, Reno, NV). Spheroids were prepared for orthotopic implantation in one well of a 96 well ultra-low attachment plate in a 100 µl 1:1 Matrigel to medium suspension. Using a 1 ml syringe and a cooled 20-gauge cannula, the six-day maturated spheroid (starting cell number: 25,000) was aspirated.