Journal: Stem Cell Research

Article Title: Testing organ-specific responses to therapies in tissues differentiated from Cystic Fibrosis patient derived iPSCs

doi: 10.1016/j.scr.2025.103653

Figure Lengend Snippet: Fig. 1. A) (Daoud and Munera, 2019; Daoud and Munera, 2020) General protocol for generating human colonic organoids (HCOs) as previously described from patient-derived iPSCs. Definitive endoderm (DE) formation was induced in iPSCs for 3 days followed by hindgut induction for 4 days. Mid-hindgut (MHE) spheroids were collected, plated in Matrigel® bubbles and incubated in colonic induction media for 3 days. Immature organoids were incubated in an intestinal growth medium until day 21, when they were switched to a growth factor conditioned media for cavity formation. B) HCO generated from WtLine#1 were stained for epithelial marker E-cadherin, intestinal epithelial marker CDX2, colonic marker SATB2 and DAPI (bottom). Images were obtained 20x magnification. Scale bar = 50 μm. C) (Jiang et al., 2021; Ahmadi et al., 2017) iPSC-derived organoids were split open as described by Xia et al. (Daoud and Munera, 2019) for subsequent fluorescence- based membrane potential assay. HCOs were removed from the Matrigel® bubble, washed and transferred into a 96-well plate to induce the split-open orga noid formation.

Article Snippet: The immature structures are patterned into colonic organoids by incubation in an intestinal growth medium (Advanced DMEM/F-12, N2, B27, 15 mM HEPES, 2 mM L-glutamine, penicillin–streptomycin) supplemented with 100 ng/mL EGF (R&D Systems, cat # 236-EG) and 100 ng/mL of BMP2 (R&D Systems, cat # 355- BM-100) for 3 days.

Techniques: Derivative Assay, Incubation, Generated, Staining, Marker, Fluorescence, Membrane