Chiron 99021

The H1 embryonic stem cell (ESC) line was directed into definitive endoderm and subsequently HIOs as previously described by our groups.^{1 (link), 4 (link)} Briefly, H1 ESCs (WiCell research Institute, Inc) were grown in feeder-free conditions in Matrigel (BD Biosciences) and cultured in mTESR1 media (Stem Cell Technologies). Upon differentiation, the ESCs were harvested and treated with Activin A to induce the formation of definitive endoderm (DE).^{5 (link)} The subsequent DE was then treated with midgut/hindgut differentiation media containing FGF4 (R&D) and Chiron 99021 (Tocris) to induce the formation of mid-hindgut spheroids. These spheroids were maintained in Mini-Gut Media with EGF (R&D) for 14 days to generate human intestinal organoids (HIOs). After 14 days, the HIOs were “split” and individually plated in Matrigel. HIOs were cultured for an additional 14 days in Mini-Gut Media and then were either processed and embedded for analysis (in vitro group) or prepared for transplantation (in vivo group). The HIOs used in this experiment were of the same batch for both the groups to ensure biological replicates of comparable size. Of note, work by our group and our collaborators has established that HIOs grown in vitro for a prolonged period do not have enhanced growth, maturation, or engraftment (data not shown).^{1 (link), 2 (link)}

Human intestinal organoids were generated and maintained as previously described (Munera and Wells, 2017 (link); Watson et al., 2014 (link)). Human embryonic stem cells and induced pluripotent stem cells were grown in feeder-free conditions in six-well Nunclon surface plates (Nunc) coated with Matrigel (BD Biosciences) and maintained in mTESR1 media (Stem Cell Technologies). For induction of definitive endoderm (DE), human ES or iPS cells were passaged with Accutase (Invitrogen) and plated at a density of 100,000 cells per well in a Matrigel-coated, Nunclon surface 24-well plate. For Accutase split cells, 10 μM Y27632 compound (Sigma) was added to the media for the first day. After the first day, media was changed to mTESR1 and cells were grown for an additional 24 hours. Cells were then treated with 100 ng/mL of Activin A for 3 days as previously described (Spence et al., 2011 (link)). DE was then treated with hindgut induction medium (RPMI 1640, 2 mM L-glutamine, 2% decomplemented FBS, penicillin-streptomycin and 100 ng/mL Activin A) for 4 d with 500 ng/mL FGF4 (R&D) and 3 μM Chiron 99021 (Tocris) to induce formation of mid-hindgut spheroids.