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For iHLO differentiation, iPSCs were dissociated with Versene (Thermo Fisher Scientific, catalog: 15040066) on Day 0 and plated into an AggreWell plate (STEMCELL Technologies, catalog: #34421) in Stemflex Medium (Thermo Fisher Scientific) to form aggregates, which were then transferred to an ultralow attachment dish on Day 1 and cultured in RPMI1640 supplemented with B27 supplement minus insulin (RPMI/B27-Insulin), CHIR99021 (1 M), Activin A (100 ng/mL), WNT3a (50 ng/mL), and Rock Inhibitor (Y27632, 5 M)

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