HIOs were generated and maintained as previously described12 (link),32 (link),33 (link).
Briefly, human H1 embryonic stem cells (WA-01; WiCell) (passage number 40–55), obtained from the Pluripotent Stem Cell Facility in our institute, were grown in feeder-free conditions in mTESR1 medium (Stem Cell Technologies). For induction of definitive endoderm, cells were split with Accutase (Invitrogen) and plated at a density between 70,000 and 100,000 cells per well in a Matrigel-coated 24-well plate. Once the cells reached 80–95% confluency, they were treated with 100 ng ml−1 Activin A for 3 d as previously described34 (link). Definitive endoderm was then treated for 4 d with hindgut induction medium containing 500 ng ml−1 FGF4 (R&D) and 3 μM Chiron 99021 (Tocris) to induce formation of mid-hindgut spheroids. Spheroids were then plated in Matrigel (Corning) and maintained in intestinal growth medium supplemented with 100 ng ml−1 EGF (R&D) to generate HIOs. Medium was changed twice weekly and HIOs were replated in fresh Matrigel at 14 d.
Briefly, human H1 embryonic stem cells (WA-01; WiCell) (passage number 40–55), obtained from the Pluripotent Stem Cell Facility in our institute, were grown in feeder-free conditions in mTESR1 medium (Stem Cell Technologies). For induction of definitive endoderm, cells were split with Accutase (Invitrogen) and plated at a density between 70,000 and 100,000 cells per well in a Matrigel-coated 24-well plate. Once the cells reached 80–95% confluency, they were treated with 100 ng ml−1 Activin A for 3 d as previously described34 (link). Definitive endoderm was then treated for 4 d with hindgut induction medium containing 500 ng ml−1 FGF4 (R&D) and 3 μM Chiron 99021 (Tocris) to induce formation of mid-hindgut spheroids. Spheroids were then plated in Matrigel (Corning) and maintained in intestinal growth medium supplemented with 100 ng ml−1 EGF (R&D) to generate HIOs. Medium was changed twice weekly and HIOs were replated in fresh Matrigel at 14 d.
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